Monomers and polymers for functional polycarbonates and poly(ester-carbonates) and PEG-co-polycarbonate hydrogels

ABSTRACT

The invention generally relates to functional polymers and hydrogels. More particularly, the invention provides versatile monomers and polymers with well-defined functionalities, e.g., polycarbonates and poly(ester-carbonates), compositions thereof, and methods for making and using the same. The invention also provides cytocompatible poly(ethylene glycol)-co-polycarobonate hydrogels (e.g., crosslinked by copper-free, strain-promoted “click” chemistry).

PRIORITY CLAIMS AND RELATED PATENT APPLICATIONS

This application is the national phase of PCT/US12/26427, filed on Feb.24, 2012, which claims the benefit of priority from U.S. ProvisionalApplication Ser. No. 61/446,997, filed on Feb. 25, 2011, and U.S.Provisional Application Ser. No. 61/484,138, filed on May 9, 2011, theentire content of which is incorporated herein by reference in itsentirety.

GOVERNMENT RIGHTS

The United States Government has certain rights to the inventionpursuant to Grant No. R01AR055615 and R01GM088678 from NationalInstitute of Health to the University of Massachusetts Medical School.

TECHNICAL FIELD OF THE INVENTION

The invention generally relates to functional polymers and hydrogels.More particularly, the invention relates to versatile monomers andpolymers with well-defined functionalities, e.g., polycarbonates andpoly(ester-carbonates), compositions thereof, and methods for making andusing the same. The invention also relates to cytocompatiblepoly(ethylene glycol)-co-polycarobonate hydrogels (e.g., crosslinked bycopper-free, strain-promoted “click” chemistry).

BACKGROUND OF THE INVENTION

Biodegradable polymers are useful in a variety of applications, forexample, in controlled drug delivery, tissue engineering and medicaldevices. Advanced biomedical applications require biomaterials to notonly provide necessary structural/mechanical support andbiodegradability over an appropriate time frame, but also to possessdefined chemical and biochemical properties to positively interact withthe living system. Biocompatible and biodegradable polymers that sharecore structural features while exhibiting incremental variations inchemical functionalities and physical properties are valuable forscreening optimal drug delivery vehicles and tissue engineeringscaffolds. (Hook, et al. Biomaterials 2010, 31, 187; Hubbell, Nat.Biotechnol. 2004, 22, 828.)

Macromolecular architectures and compositions with a range of mechanicalproperties and degradation profiles have been reported. (Nair, et al.Prog. Polym. Sci. 2007, 32, 762; Sodergard, et al. Prog. Polym. Sci.2002, 27, 1123.) For example, aliphatic polycarbonates have attractedincreasing interest due to their non-acidic degradation products andpotential to introduce properties complementary to those obtainable byother degradable polymers. (Pego, et al. J. Biomater. Sci., Polym. Ed.2001, 12, 35; Pego, et al. Macromol. Biosci. 2002, 2, 411; Rokicki,Prog. Polym. Sci. 2000, 25, 259; Bat, et al. Biomaterials 2010, 31,8696; Dankers, et al. Macromolecules 2006, 39, 8763; Zhu, et al.Macromolecules 1991, 24, 1736.) The hydrophobic nature and the lack ofside chain functionalities of polycarbonates, however, have limitedtheir biomedical applications. (Zelikin, et al. Biomacromolecules 2006,7, 3239.) Indeed, such limitations are shared by synthetic biodegradablepolymers in general, including polyesters, polyanhydrides, andpolyorthoesters. (Vert, Biomacromolecules 2005, 6, 538; Rasal, et al.Prog. Polym. Sci. 2010, 35, 338; Kumar, et al. Adv. Drug Delivery Rev.2002, 54, 889.)

Current methods for imparting functionalities and improving thehydrophilicity of biodegradable polymers include post-polymerizationsurface irradiation grafting, post-polymerization end-groupmodification, polymerization initiated by hydrophilic/functional polymerprecursors, and (co)polymerization of functional monomers. (Edlund, etal. J. Am. Chem. Soc. 2005, 127, 8865; Suriano, et al. J. Polym. Sci.,Part A: Polym. Chem. 2010, 48, 3271; He, et al. Biomacromolecules 2006,7, 252; Zhang, et al. J. Controlled Release 2006, 112, 57; Gautier, etal. J. Biomater. Sci., Polym. Ed. 2003, 14, 63; Lu, et al.Macromolecules 2010, 43, 4943; Trollsas, et al. Macromolecules 2000, 33,4619; Jiang, et al. Abstr. Papers Am. Chem. Soc. 2005, 230, U4073;Gerhardt, et al. Biomacromolecules 2006, 7, 1735; Hu, et al.Biomacromolecules 2008, 9, 553; Pratt, et al. Chem. Commun. 2008, 114;Hu, et al. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 7022; Hu, etal. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 5518; Xie, et al. J.Polym. Sci., Part A: Polym. Chem. 2007, 45, 1737; Lou, et al. Macromol.Rapid Commun. 2003, 24, 161; Detrembleur, et al. Macromolecules 2000,33, 14; Rieger, et al. Macromolecules 2004, 37, 9738; Yin, et al.Macromolecules 1999, 32, 7711.)

(Co)Polymerization of functional monomers provides a straightforward wayto introduce functionalities and hydrophilicity with better-controlledpolymer compositions and structures provided that suitable monomerscould be designed. (Trollsas, et al. Macromolecules 2000, 33, 4619;Gerhardt, et al. Biomacromolecules 2006, 7, 1735.) However, due to theincompatibility of most reactive groups (e.g. hydroxyls, amines,carboxyls, and thiols) with the polymerization conditions, cumbersomeprotection and post-polymerization deprotection steps involving heavymetal catalysts and lowering overall yields are often required.(Trollsas, et al. Macromolecules 2000, 33, 4619; Hu, et al.Biomacromolecules 2008, 9, 553; Vandenberg, et al. Macromolecules 1999,32, 3613; Zhang, et al. Macromolecules 2009, 42, 1010; Noga, et al.Biomacromolecules 2008, 9, 2056; Hu, et al. J. Polym. Sci., Part A:Polym. Chem. 2008, 46, 7022; Sanda, et al. Macromolecules 2001, 34,1564; Kimura, et al. Macromolecules 1988, 21, 3338; Al-Azemi, et al.Macromolecules 1999, 32, 6536; Pounder, et al. Biomacromolecules 2010,11, 1930.) The degradable nature of the backbone of these polymers alsoimposes additional challenges to the preparation of well-definedfunctional derivatives.

Although several monomers with “clickable” functionalities includingalkyne- and (methyl)acrylate-containing lactones or carbonates have beenreported, a pressing need exists for monomers functionalized withreactive groups orthogonal to polymerization conditions that enablefacile post-polymerization functionalization without tediousprotection/deprotection. (Parrish, et al. J. Am. Chem. Soc. 2005, 127,7404; Han, et al. Macromol. Biosci. 2008, 8, 638; Jiang, et al.Macromolecules 2008, 41, 1937; Darcos, et al. Polymer Chemistry 2010, 1,280; van der Ende, et al. Macromolecules 2010, 43, 5665; Chen, et al.Macromolecules 2010, 43, 201; Iha, et al. Chem. Rev. 2009, 109, 5620;Sumerlin, et al. Macromolecules 2010, 43, 1.)

Biocompatible hydrogels are important materials in biomedical researchand pharmaceutical products. (Jen, et al. Biotechnol. Bioeng. 1996, 50,357; Wang, et al. Adv. Drug Delivery Rev. 2010, 62, 699; Gkioni, et al.Tissue Eng. Part B-Rev 2010, 16, 577; Hynd, et al. Biomater. Sci.,Polym. Ed. 2007, 18, 1223; Ifkovits, et al. Tissue Eng. 2007, 13, 2369;Khetan, et al. Soft Matter 2011, 7, 830; Kim, et al. Tissue Engineeringand Regenerative Medicine 2011, 8, 117; Lee, et al. Chem. Rev. 2001,101, 1869.) Biocompatible hydrogels have been used as proteinmicrochips, drug and gene delivery carriers, ophthalmic prostheses, andscaffolds for encapsulating cells to facilitate either the investigationof cell-extracellular matrix interactions or tissue regenerations.(Bertone, et al. FEBS J. 2005, 272, 5400; Hoare, et al. Polymer 2008,49, 1993; Alvarez-Lorenzo, et al. J. Drug Deliv. Sci. Tec. 2010, 20,237; Drury, et al. Biomaterials 2003, 24, 4337; Tessmar, et al. Adv.Drug Delivery Rev. 2007, 59, 274; Burdick, et al. Adv. Mater. 2011, 23,H41; Minh, et al. Macromolecular Bioscience 2010, 10, 563; Marklein, etal. Adv. Mater. 2010, 22, 175; Anderson, et al. Biomaterials 2011, 32,3564; Benoit, et al. Nat. Mater. 2008, 7, 816; Haines-Butterick, et al.Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 7791; Chung, et al. TissueEngineering Part A 2009, 15, 243; Annabi, et al. Tissue Eng. Part B-Rev2010, 16, 371.) Naturally occurring biopolymers such as collagens,fibrin, alginate, agarose, hyaluronan and chondroitin sulfate, as wellas synthetic polymers such as polyethylene glycol) (PEG), polyvinylalcohol) (PVA), poly(N-isopropylacrylamine) (PNIPAAM) have been used forregenerative medicine applications. (Drury, et al. Biomaterials 2003,24, 4337.)

The chemistry, microstructure and physical properties of hydrogel tissuescaffolds have significant influences on the fate of their residentcells. (Lutolf, et al. Nature 2009, 462, 433; Even-Ram, et al. Cell2006, 126, 645; Cushing, et al. Science 2007, 316, 1133.) Synthetichydrogels present unique advantages over naturally occurring hydrogelsdue to the broader tunability of the properties of the former. (Kloxin,et al. Science 2009, 324, 59; Lee, et al. Biomaterials 2006, 27, 5268;Luo, et al. Nature Materials 2004, 3, 249.) Challenges still exist,however, for the translation of existing synthetic hydrogels forbiomedical uses. For instance, the gelling of most physicallycrosslinked hydrogels requires substantial changes in environmentalconditions (e.g., pH, temperature, ionic strength), which can bedetrimental to the in situ encapsulated cells. In addition, theintegrity of these physically crosslinked cell-gel constructs aredifficult to maintain in vivo. On the other hand, the cytotoxicity ofcrosslinking reagents and initiators used for chemically crosslinkedhydrogels can negatively impact the viability and long-term fate of theencapsulated cells. (Mann, et al. Biomaterials 2001, 22, 3045;Rouillard, et al. Tissue Engineering Part C-Methods 2011, 17, 173; Shu,et al. Biomaterials 2003, 24, 3825.)

In general, chemical crosslinking conditions and chemically crosslinkednetworks deemed cyto-compatible are still limited. (Hennink, et al. Adv.Drug Delivery Rev. 2002, 54, 13.) Among them, PEG-based hydrogels formedby photo-initiated radical polymerization of (meth)acrylated PEGmacromers have been the most utilized for the encapsulation and supportof tissue-specific differentiation of stem cells. Major limitationsassociated with photo-crosslinked PEG gels include the intrinsicheterogeneities of the network structures due to the uncontrolledradical polymerization process and the varied degrees of cytotoxicity ofthe aqueous radical initiators utilized (e.g., 1-2959 and VA-086).(Rouillard, et al. Tissue Engineering Part C-Methods 2011, 17, 173.)Alternative in situ crosslinking strategies involving disulfide bondformations or Michael addition reactions between thiols and acryaltes orvinyl sulfones can eliminate the need for radical initiators, but stillsuffer from the potential interference from the thiol residues widelypresent within the tissue environment.

Thus, a hydrogel system that can be crosslinked under physiologicalconditions without external perturbations or cross-reactivities withcellular or tissue environment is highly desired. For tissueregeneration applications, the hydrogels should also ideally possessadequate mechanical properties and exhibit tunable degradation ratespotentially matching with those of the tissue integrations.

SUMMARY OF THE INVENTION

The invention is based, in part, on certain novel carbonate-basedmonomers, their controlled homopolymerization and copolymerization, andthe facile functionalization of the resulting polycarbonates andpoly(ester-carbonates), for example, via copper-catalyzed (CuAAC) andstrain-promoted (SPAAC) azido-alkyne cycloaddition “click” chemistries.(Kolb, et al. Angew. Chem., Int. Ed. 2001, 40, 2004; Agard et al. J. Am.Chem. Soc. 2004, 126, 15046.)

In one aspect, the invention generally relates to a compound having thestructural formula of:

wherein, each of R₁, R₂, R₃, R₄, R₅ and R₆ independently is hydrogen, analkyl, an alkoxy, —OH, a halide, an aryl, an aryloxy, an acyl, ahaloalkyl group, provided that one or more of R₁, R₂, R₃, R₄, R₅ and R₆independently is R_(x), wherein R_(x) is a group that comprises eitheran azide group or an alkynyl group.

In certain preferred embodiments, R_(x) is:

L_(N)-N₃,

L_(C)-≡-R_(C), or

L_(N)-R_(y)—R₉,wherein L_(N) is a linking group, L_(C) is a linking group, R_(C) is ahydrogen or a linear or branched, unsubstituted or substituted alkylgroup, R_(y) is a coupling moiety, and R₉ is a pendant group, and one ormore of the carbon atom may be optionally substituted with a hetero-atomselected from O, S and N or with a —(C═O)— group.

In certain preferred embodiments, two of R₁, R₂, R₃, R₄, R₅ and R₆ areindependently R_(x) and the rest are hydrogen, for example, wherein R₃and R₄ are R_(x):

In certain preferred embodiments, one of R₁, R₂, R₃, R₄, R₅ and R₆ isR_(x) and the rest are hydrogen, for example, where R₃ is R_(x):

In another aspect, the invention generally relates to a monomer unithaving the structure of:

wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ independently in hydrogen, analkyl, an alkoxy, —OH, a halide, an aryl, an aryloxy, an acyl, ahaloalkyl, provided that one or more of R₁, R₂, R₃, R₄, R₅ and R₆independently is independently R_(x), wherein R_(x) is a group thatcomprises either an azide group or an alkynyl group.

In certain preferred embodiments, two of R₁, R₂, R₃, R₄, R₅ and R₆independently is R_(x), for example,

In certain preferred embodiments, one of R₁, R₂, R₃, R₄, R₅ and R₆ isR_(x), for example,

In yet another aspect, the invention generally relates to a polymercomprising a structural unit of:

wherein each of R₁, R₂, R₃, R₄, R₅, and R₆ independently in hydrogen, analkyl, an alkoxy, —OH, a halide, an aryl, an aryloxy, an acyl, ahaloalkyl group, provided that one or more of R₁, R₂, R₃, R₄, R₅, and R₆independently is independently R_(x), wherein R_(x) is a group thatcomprises either an azide group or an alkynyl group; and n is an integerfrom about 1 to about 2,000 (e.g., 1, 5, 10, 20, 50, 100, 200, 500,1000, 2000).

In yet another aspect, the invention generally relates to a co-polymercomprising the monomer units of:

wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ independently in hydrogen, analkyl, an alkoxy, —OH, a halide, an aryl, an aryloxy, an acyl, ahaloalkyl group, provided that one or more of R₁, R₂, R₃, R₄, R₅ and R₆independently is independently R_(x), wherein R_(x) is a group thatcomprises either an azide group or an alkynyl group; each of R₇ and R₈is independently hydrogen or an alkyl group; and n is an integer, forexample, from about 1 to about 16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16).

In yet another aspect, the invention generally relates to a method forpreparing a hydrogel of poly(ethylene glycol)-co-polycarbonate. Themethod includes: (1) providing a first poly(ethylene glycol) macromercomprising a poly(ethylene glycol) oligomer segment and a flankingsegment on one or more side thereof, wherein each flanking segmentcomprises a single- or multi-azido-functionalized biodegradablepolycarbonate block; (2) providing a second poly(ethylene glycol)macromer comprising a poly(ethylene glycol) oligomer segment and aflanking segment on one or more side thereof, wherein each flankingsegment comprises a terminal alkyne group; and (3) forming a hydrogel bycrosslinking the first macromer and the second macromer under conditionsso as to effect azide-alkyne cycloaddition.

In certain preferred embodiments, the method further includes: providingcells prior to the crosslinking step; and crosslinking the firstmacromer and the second macromer in the presence of cells underconditions so as to effect copper-free, strain-promoted azide-alkynecycloaddition.

In certain preferred embodiments, the first poly(ethylene glycol)macromer has the structural formula of:

wherein each R is a group that comprises either an azide group or analkynyl group, and n is an integer from about 0 to about 5,000 (e.g., 0,1, 5, 10, 20, 50, 100, 500, 1,000, 2,000, 5,000). For example,each R may independently be

wherein each of R₃ and R₄ independently in hydrogen, an alkyl, analkoxy, —OH, a halide, an aryl, an aryloxy, an acyl, a haloalkyl group,provided that one or both of R₃ and R₄ independently is independentlyR_(x), wherein R_(x) is a group that comprises either an azide group oran alkynyl group, and n is an integer from about 1 to about 2,000 (e.g.,1, 5, 10, 20, 50, 100, 200, 500, 1000, 2000).

In certain embodiments, the second poly(ethylene glycol) macromer hasthe general structural formula of:

wherein R is

and each of a, b, c, and d is an integer and from about 1 to about 2000(e.g., 1, 5, 10, 20, 50, 100, 200, 500, 1000, 2000).

In yet another aspect, the invention generally relates to a hydrogelcomposition that includes a hydrogel composition comprising acrosslinked product of a first macromer and a second macromere. Thefirst macromer comprises a water-soluble hydrophilic polymer segment anda flanking segment on one or more side thereof, wherein each flankingsegment comprises a single- or multi-azido-functionalized biodegradablepolycarbonate block. The second macromer comprises a water-solublehydrophilic polymer segment and a flanking segment on one or more sidethereof, wherein each flanking segment comprises a terminal alkynegroup.

In certain preferred embodiments, the first macromer is a firstpoly(ethylene glycol) macromer and the second macromer is a secondpoly(ethylene glycol) macromere. The first poly(ethylene glycol)macromer comprises a poly(ethylene glycol) oligomer segment and aflanking segment on one or more side thereof, wherein each flankingsegment comprises a single- or multi-azido-functionalized biodegradablepolycarbonate block. The second poly(ethylene glycol) macromer comprisesa poly(ethylene glycol) oligomer segment and a flanking segment on oneor more side thereof, wherein each flanking segment comprises a terminalalkyne group.

In yet another aspect, the invention generally relates to acytocompatible hydrogel composition suitable for use in tissue repair orregeneration, comprising a three-dimensional construct of cells and acrosslinked network of a first poly(ethylene glycol) macromer and asecond poly(ethylene glycol) macromere. The first poly(ethylene glycol)macromer comprises a poly(ethylene glycol) oligomer segment and aflanking segment on one or more side thereof, wherein each flankingsegment comprises a single- or multi-azido-functionalized biodegradablepolycarbonate block. The second poly(ethylene glycol) macromer comprisesa poly(ethylene glycol) oligomer segment and a flanking segment on oneor more side thereof, wherein each flanking segment comprises a terminalalkyne group.

The invention encompasses any product that is comprised of a polymer orco-polymer disclosed herein including any product prepared from apolymer or co-polymer of disclosed herein such as by post-polymerizationfunctionalization. The invention also encompasses any product that iscomprised of a hydrogel (with or without encapsulated cells) disclosedherein including any product prepared from a hydrogel disclosed hereinsuch as by post-polymerization functionalization.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthesis and polymerization kinetics ofazido-functionalized cyclic carbonate monomer AzDXO.

FIG. 2 shows the differential scanning calorimetry of homopolymers ofAzDXO with different molecular weights.

FIG. 3 shows the preparation of block and random copolymers of L-Lactide(L-LA) and AzDXO by sequential and one-pot polymerization, respectively.i) 0.01M DBU, CHCl₃, rt.

FIG. 4 shows GPC traces and DSC scans confirming the successfulpreparation of block and random copolymers of L-LA and AzDXO.

FIG. 5 shows the functionalization of block and random copolymers with5-hexyn-1-ol (HX) via copper-catalyzed azido-alkyne cycloaddition(CuAAC).

FIG. 6 shows representative GPC traces and ¹H NMR spectra indicating thesuccessful modification of block and random copolymers bystrain-promoted azido-alkyne cycloaddition (SPAAC).

FIG. 7 shows representative ¹H NMR monitoring of the homopolymerizationof AzDXO in CDCl₃ at room temperature using benzyl alcohol (Bz) as theinitiator and 0.01M DBU as the catalyst.

FIG. 8 shows representative DSC scans of random copolymers of AzDXO andL-Lactide with different compositions.

FIG. 9 shows representative ¹H NMR spectra of (a) homopolymerPyrene-P(AzDXO₁₀₀), (b) block copolymer Pyrene-P(LLA₁₀₀)-b-P(AzDXO₂₀),and (c) random copolymer Pyrene-P(LLA₁₀₀-co-AzDXO₂₀).

FIG. 10 shows representative ¹³C NMR spectra of (a) homopolymerPyrene-P(AzDXO₁₀₀), (b) block copolymer Pyrene-P(LLA₁₀₀)-b-P(AzDXO₂₀),and (c) random copolymer Pyrene-P(LLA₁₀₀-co-AzDXO₂₀).

FIG. 11 shows representative DSC scans of block copolymers with fixedL-lactide block length but varying AzDXO block lengths.

FIG. 12 shows representative FTIR spectra of random and block copolymersbefore and after modification using 5-hexyn-1-ol (HX) through CuAACsupported quantitative reaction.

FIG. 13 shows representative ¹H NMR spectra confirming the successfulattachment of 5-hexyn-1-ol (HX) (a) to the block copolymerPyrene-P(LLA₁₀₀)-b-P(AzDXO₂₀)-HX (b) and the random copolymerPyrene-P(LLA₁₀₀-co-AzDXO₂₀)-HX (c).

FIG. 14 shows representative ¹H NMR of2-(bromomethyl)-2-methylpropane-1,3-diol (BMMPDiol) in CDCl₃.

FIG. 15 shows representative ¹³C NMR of2-(bromomethyl)-2-methylpropane-1,3-diol (BMMPDiol) in CDCl₃.

FIG. 16 shows representative ¹H NMR of2-(azidomethyl)-2-methylpropane-1,3-diol (AMMPDiol) in CDCl₃.

FIG. 17 shows representative ¹³C NMR of2-(azidomethyl)-2-methylpropane-1,3-diol (AMMPDiol) in CDCl₃.

FIG. 18 shows representative ¹H NMR of5-(azidomethyl)-5-methyl-1,3-dioxan-2-one (AMMDXO) in CDCl₃.

FIG. 19 shows representative ¹³C NMR of5-(azidomethyl)-5-methyl-1,3-dioxan-2-one (AMMDXO) in CDCl₃.

FIG. 20 shows representative ¹H NMR of MPEG2k-AMMDXO in CDCl₃.

FIG. 21 shows representative ¹³C NMR of MPEG2k-AMMDXO in CDCl₃.

FIG. 22 shows macromer synthesis, crosslinking and cell encapsulationstrategies: a) Ring-opening polymerization (ROP) of AzDXO initiated byPEG. [AzDXO]=0.1125 M, [DBU]=0.1 M, rt, Ar, 4 h; b) Synthesis ofPEG-(DBCO)_(x) by reacting DBCO-acid with PEG in CH₂Cl₂ under thecatalysis of DIPC and DPTS, rt, 10 h; c) Depiction of the cellencapsulation by crosslinking PEG-P(AzDXO)_(2m) and PEG-(DBCO)_(x) viaSPAAC “click” reaction; d) A representative demonstration of the rapidgellation of the cell-hydrogel constructs within 1 min of mixing theBMSC cell suspension (10⁶ cells/mL) in a PEG20k-P(AzDXO)₄ solution (10w/v % in expansion media) and a 4-arm-PEG10k-DBCO solution (10 w/v % inexpansion). The BMSC expansion media consisted of α-MEM without ascorbicacid and 20% FBS.

FIG. 23 shows representative ¹H NMR spectra and proton integrations ofPEG6k-P(AzDXO)₄ (bottom) supporting successful polymerization of AzDXO(top) initiated from the end hydroxyl groups of PEG6k. Protonintegrations support a copolymer composition approximating that of thetheoretical estimate.

FIG. 24 shows representative ¹H NMR spectra and proton integrations ofPEG20k-(DBCO)₂ (bottom) supporting the successful attachment ofDBCO-acid (top) on both ends of PEG20k.

FIG. 25 shows time-dependent shear storage moduli (G′, black symbols)and shear loss moduli (G″, blue symbols) of the various hydrogelformulations during the SPAAC crosslinking.

FIG. 26 shows viability of BMSC cells encapsulated by the “click”hydrgles. (a) Representative confocal Z-stack (400 μm) images ofencapsulated BMSC cells stained with a live/dead viability staining kit24 h after initial encapsulation. Live cells were stained green whiledead cells were stained red; (b) MTT cell viability assay performed onthe hydrogel-BMSC constructs (10⁶ cells/mL) 48 h after cellencapsulation showing better cell viability for all “click”hydrogel-encapsulated BMSCs than those encapsulated in photocrosslinkedPEG6k-DMA hydrogels. * indicates p<0.05 (student t-test) between the“click” gel and the PEG6k-DMA control (crosslinked in the media).

FIG. 27 shows representative ¹³C NMR of 4-arm-PEG10k-(DBCO)₄ showing thedisappearance of the characteristic peak for —CH₂OH endgroups of4-arm-PEG10k from 61.4 ppm, supporting complete esterification of —CH₂OHby DBCO-acid.

FIG. 28 shows MTT viability assay of BMSC cells cultured on 96-welltissue culture plates in the presence of PEG-P(AzDXO)_(2m) andPEG-(DBCO)_(x) macromers showing cell viability at 48 h comparable tothose cultured in the absence of any macromers. For cell seeding, BMSCcell suspension (10⁶ cells/mL, 50 μL) in expansion media (α-MEM withoutascorbic acid, 20% FBS) containing 0 or 10 w/v % macromers was added toeach well of the 96-well plate before an extra 200 μL of expansion mediawas added. No statistically significant difference was observed betweenany of the macromer-treated cultures and the no-macromer control culture(p>0.05; student t-test).

DEFINITIONS

Definitions of specific functional groups and chemical terms aredescribed in more detail below. General principles of organic chemistry,as well as specific functional moieties and reactivity, are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

Given the benefit of this disclosure, one of ordinary skill in the artwill appreciate that synthetic methods, as described herein, may utilizea variety of protecting groups. By the term “protecting group”, as usedherein, it is meant that a particular functional moiety, e.g., O, S, orN, is temporarily blocked so that a reaction can be carried outselectively at another reactive site in a multifunctional compound. Inpreferred embodiments, a protecting group reacts selectively in goodyield to give a protected substrate that is stable to the projectedreactions; the protecting group should be selectively removable in goodyield by preferably readily available, non-toxic reagents that do notattack the other functional groups; the protecting group forms an easilyseparable derivative (more preferably without the generation of newstereogenic centers); and the protecting group has a minimum ofadditional functionality to avoid further sites of reaction. Oxygen,sulfur, nitrogen, and carbon protecting groups may be utilized. Examplesof a variety of protecting groups can be found in Protective Groups inOrganic Synthesis, Third Ed. Greene, T. W. and Wuts, P. G., Eds., JohnWiley & Sons, New York: 1999.

It will be appreciated that the compounds, monomers and polymers, asdescribed herein, may be substituted with any number of substituents orfunctional moieties.

The term (C_(x)-C_(y)), as used herein, refers in general to groups thathave from x to y (inclusive) carbon atoms. Therefore, for example, C₁-C₆refers to groups that have 1, 2, 3, 4, 5, or 6 carbon atoms, whichencompass C₁-C₂, C₁-C₃, C₁-C₅, C₂-C₃, C₂-C₄, C₂-C₅, C₂-C₆, and all likecombinations. (C₁-C₂₀) and the likes similarly encompass the variouscombinations between 1 and 20 (inclusive) carbon atoms, such as (C₁-C₆),(C₁-C₁₂) and (C₃-C₁₂).

The term “acyl,” as used herein, refers to alkanoyl group —C(═O)R_(d),where R_(d) is alkyl, alkenyl, alkynyl, aryl or aralkyl.

The term “alkoxy,” as used herein, refers to the ether-O-alkyl, whereinalkyl is defined herein. The term “(C_(x)-C_(y))alkoxy” refers to astraight or branched chain alkyl group consisting essentially of from xto y carbon atoms that is attached to the main structure via an oxygenatom, wherein x is an integer from 1 to about 10 and y is an integerfrom about 2 to about 20. For example, “(C₁-C₂₀)alkoxy” refers to astraight or branched chain alkyl group having 1-20 carbon atoms that isattached to the main structure via an oxygen atom, thus having thegeneral formula alkyl-O—, such as, for example, methoxy, ethoxy,propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentoxy,2-pentyl, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, and3-methylpentoxy.

The term “alkyl,” as used herein, refers to saturated aliphatic groupsincluding straight chain, branched chain, and cyclic groups all of themmay be optionally substituted. As used herein, the term“(C_(x)-C_(y))alkyl” refers to a saturated linear or branched freeradical consisting essentially of x to y carbon atoms, wherein x is aninteger from 1 to about 10 and y is an integer from about 2 to about 20.Exemplary (C_(x)-C_(y))alkyl groups include “(C₁-C₂₀)alkyl,” whichrefers to a saturated linear or branched free radical consistingessentially of 1 to 20 carbon atoms and a corresponding number ofhydrogen atoms. Other examples include (C₁-C₆)alkyl, (C₁-C₁₂)alkyl(C₁-C₁₆)alkyl groups. Preferred alkyl groups contain 1 to 16 carbonatoms (e.g., “1-12 carbon atoms”). Suitable alkyl groups include methyl,ethyl and the like, and may be optionally substituted.

The term “alkynyl,” as used herein, refers to unsaturated hydrocarbongroups which contain at least one carbon-carbon triple bond and includesstraight chain and branched chain groups which may be optionallysubstituted. Preferred alkynyl groups have two to eighteen carbon atoms.Preferable alkynyl groups have two to twelve carbons. Suitable alkynylgroups include ethylnyl, propynyl and the like, and may be optionallysubstituted.

The term “amino,” as used herein, refers to —NR_(a)R_(b), where R_(a)and R_(b) are independently hydrogen, lower alkyl or an acyl group.

The term “aryl,” as used herein, refers to aromatic groups which have atleast one ring having a conjugated pi electron system and includescarbocyclic aryl, biaryl, both of which may be optionally substituted.As used herein, the term “(C_(x)-C_(y))aryl” refers to an aromatic groupconsisting essentially of x to y carbon atoms in the aromatic ring(s),wherein x is an integer from about 6 to about 10 and y is an integerfrom about 10 to about 14. For example, “(C₆-C₁₀)aryl” refers to anaromatic group consisting essentially of 6 to 10 ring carbon atoms,e.g., phenyl and naphthyl. Preferred aryl groups have 6 to 10 carbonatoms. Suitable aryl groups include phenyl and napthyl.

The term “aryloxy,” as used herein, refers to the ether-O-aryl, whereinaryl is defined herein.

The term “azide,” as used herein, refers to the group —N₃ (or —N═N⁺═N⁻).

The term “haloalkyl,” as used herein, refers to an alkyl substitutedwith one or more halogens.

The term “hydrocarbyl”, as used herein, refers to a group primarilycomposed of hydrogen and carbon atoms and is bonded to the remainder ofthe molecule via a carbon atom, but it does not exclude the presence ofother atoms or groups in a proportion insufficient to detract from thesubstantial hydrocarbon characteristics of the group. The hydrocarbylgroup may be composed of only hydrogen and carbon atoms, for example, analiphatic group such as alkyl or alkylene group groups, which may belinear or branched.

The term “optionally substituted” or “substituted,” as used herein andunless otherwise specifically defined herein, refers to groupssubstituted by one to five substituents, independently selected fromlower alkyl (acyclic and cyclic), aryl, alkenyl, akynyl, alkoxy, halo,haloalkyl, amino, aminoalkyl, mercapto, alkylthio, nitro, alkanoyl,alkanoyloxy, alkanoyloxyalkanoyl, alkoxycarboxy, carbalkoxy,carboxamido, formyl, carboxy, hydroxy, cyano, azido, keto and cyclicketals thereof, alkanoylamido and hemisuccinate ester salts.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, in part, on the unexpected discovery andsyntheses of novel carbonate-based monomers (e.g., azido-substitutedcyclic trimethylene carbonate-based monomers) their controlledhomopolymerization and copolymerization (e.g., with lactide), and thefacile functionalization of the resulting polycarbonates andpoly(ester-carbonates), for example, via copper-catalyzed (CuAAC) andstrain-promoted (SPAAC) azido-alkyne cycloaddition “click” chemistries.The invention is also based, in part, on the unexpected discovery ofnovel and efficient methods for preparing cytocompatible poly(ethyleneglycol)-co-polycarbonate hydrogels, for example, crosslinked bycopper-free, strain-promoted “click” chemistry.

Monomers and Polymers for Well-Defined Functional Polycarbonates andPoly(Ester-Carbonates)

The invention enables the design, syntheses and application of versatilefunctional monomers that may be utilized for the preparation offunctional polycarbonates and functional block and random poly(estercarbonate) copolymers. These polymers meet the requirements of (1) thatthe preparation of functional monomers in large scale with high puritywithout the need for chromatographic purifications, (2) that thereactive handles (in either protected or unmasked forms) of the monomersare compatible with the downstream polymerization conditions while thedeprotection after polymerization can be carried out quantitativelyunder mild conditions if needed, and (3) that the accessibility andreactivity of the reactive handles of the monomers forpost-polymerization modifications are high, ensuring efficient andquantitative functionalization. For example, the azido-substitutedcyclic trimethylene carbonate monomers are readily polymerized toprovide biodegradable polymers with pendent azido groups, which may beused as handles to introduce a wide range of functional groups viaHuisgen 1,3-dipolar cycloaddition “click” chemistry. Other examples ofhigh efficiency and fidelity “click” chemistry include Diels-Alderreaction, Michael addition, and thiol-ene reaction. Polymers (includingcopolymers) of the invention are useful in a variety of applications,for example, in tissue engineering, drug delivery, dental devices,sutures, vascular stents and other medical devices.

In one aspect, the invention generally relates to a compound having thestructural formula of:

wherein, each of R₁, R₂, R₃, R₄, R₅ and R₆ independently is hydrogen, analkyl, an alkoxy, —OH, a halide, an aryl, an aryloxy, an acyl, ahaloalkyl group, provided that one or more of R₁, R₂, R₃, R₄, R₅ and R₆independently is R_(x), wherein R_(x) is a group that comprises eitheran azide group or an alkynyl group.

In certain preferred embodiments, R_(x) is:

L_(N)-N₃,

L_(C)-≡-R_(C), or

L_(N)-R_(y)—R₉,wherein L_(N) is a linking group, L_(C) is a linking group, R_(C) is ahydrogen or a linear or branched, unsubstituted or substituted alkylgroup, R_(y) is a coupling moiety, and R₉ is a pendant group, and one ormore of the carbon atom may be optionally substituted with a hetero-atomselected from O, S and N or with a —(C═O)— group.

In some embodiments, L_(N) is a bivalent linear or branched,unsubstituted or substituted alkylene group, wherein one or more of thecarbon atoms may be optionally substituted with a hetero-atom selectedfrom O, S and N or with a —(C═O)— group.

In certain preferred embodiments, two of R₁, R₂, R₃, R₄, R₅ and R₆ areindependently R_(x) and the rest are hydrogen, for example, wherein R₃and R₄ are R_(x):

In certain preferred embodiments, one of R₁, R₂, R₃, R₄, R₅ and R₆ isR_(x) and the rest are hydrogen, for example, where R₃ is R_(x):

In certain preferred embodiments, each R_(x) group independently is

L_(N)-N₃.

Each L_(N) may be independently a bivalent —(CH₂)_(p)— radical, whereineach p is independently an integer, for example, from about 1 to about16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16). EachL_(N) may also be independently a bivalent —(O—CH₂)_(q)— radical,wherein each q is independently an integer, for example, from about 1 toabout 16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16).

In certain preferred embodiments, each R_(x) independently is

L_(C)-≡-R_(C)wherein L_(C) and R_(C) each may be a C₁-C₁₆ alkyl group.

In certain preferred embodiments, R_(x) is:

L_(N)-R_(y)—R₉wherein each L_(N) is independently a bivalent —(CH₂)_(p)— radical,wherein each p is independently an integer, for example, from about 1 toabout 16. For example, R_(y) may be

and R₉ can be

wherein n is an integer, for example, from about 0 to about 400 (e.g.,0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400).

R_(y) may also be

wherein Q is a substituted or unsubstituted cyclic group. Q may be aC₃-C₁₂ cyclic group, optionally with one or more of the carbon atoms maybe optionally substituted with a hetero-atom selected from O, S and N orwith a —(C═O)— group. For example, Q may have the structural formula of:

wherein each of R_(Q1), R_(Q2), R_(Q3), R_(Q4) and R_(Q5) isindependently a hydrogen, a halide, —OH, or a hydrocarbyl group and mayoptionally form one or more fused rings and X is carbon or a hetero-atomselected from O, S and N or a —(C═O)— group.

In some preferred embodiments, Q has the structural formula of:

wherein each of R_(Q5), R_(Q6) and R_(Q7) is independently a hydrogen, ahalide, —OH, or a hydrocarbyl group.

In some preferred embodiments, Q has a structural formula selected from:

Other examples of Q include:

In another aspect, the invention generally relates to a monomer unithaving the structure of:

wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ independently in hydrogen, analkyl, an alkoxy, —OH, a halide, an aryl, an aryloxy, an acyl, ahaloalkyl, provided that one or more of R₁, R₂, R₃, R₄, R₅ and R₆independently is independently R_(x), wherein R_(x) is a group thatcomprises either an azide group or an alkynyl group.

In certain preferred embodiments, two of R₁, R₂, R₃, R₄, R₅ and R₆independently is R_(x), for example,

In certain preferred embodiments, one of R₁, R₂, R₃, R₄, R₅ and R₆ isR_(x), for example,

In yet another aspect, the invention generally relates to a polymercomprising a structural unit of:

wherein each of R₁, R₂, R₃, R₄, R₅, and R₆ independently in hydrogen, analkyl, an alkoxy, —OH, a halide, an aryl, an aryloxy, an acyl, ahaloalkyl group, provided that one or more of R₁, R₂, R₃, R₄, R₅, and R₆independently is independently R_(x), wherein R_(x) is a group thatcomprises either an azide group or an alkynyl group; and n is an integerfrom about 1 to about 2,000 (e.g., 1, 5, 10, 20, 50, 100, 200, 500,1000, 2000).

In certain preferred embodiments, R_(x) is:

L_(N)-N₃,

L_(C)-≡-R_(C), or

L_(N)-R_(y)—R₉,wherein L_(N) is a linking group, L_(C) is a linking group, R_(C) is ahydrogen or a linear or branched, unsubstituted or substituted alkylgroup, R_(y) is a coupling moiety, and R₉ is a pendant group, and one ormore of the carbon atom may be optionally substituted with a hetero-atomselected from O, S and N or with a —(C═O)— group.

In certain preferred embodiments, two of R₁, R₂, R₃, R₄, R₅ and R₆independently is R_(x), for example,

In certain preferred embodiments, one of R₁, R₂, R₃, R₄, R₅ and R₆ isR_(x), for example,

In yet another aspect, the invention generally relates to a co-polymercomprising the monomer units of:

wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ independently in hydrogen, analkyl, an alkoxy, —OH, a halide, an aryl, an aryloxy, an acyl, ahaloalkyl group, provided that one or more of R₁, R₂, R₃, R₄, R₅ and R₆independently is independently R_(x), wherein R_(x) is a group thatcomprises either an azide group or an alkynyl group; each of R₇ and R₈is independently hydrogen or an alkyl group; and n is an integer, forexample, from about 1 to about 16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16).

In certain preferred embodiments, R_(x) is:

L_(N)-N₃,

L_(C)-≡-R_(C), or

L_(N)-R_(y)—R₉,wherein L_(N) is a linking group, L_(C) is a linking group, R_(C) is ahydrogen or a linear or branched, unsubstituted or substituted alkylgroup, R_(y) is a coupling moiety, and R₉ is a pendant group, and one ormore of the carbon atom may be optionally substituted with a hetero-atomselected from O, S and N or with a —(C═O)— group.

The co-polymer may be a random co-polymer.

In certain embodiments, the co-polymer is a block co-polymer comprisingblocks of:

wherein m is an integer, for example, from about 1 to about 500 (e.g.,1, 3, 5, 10, 20, 50, 100, 200, 500). n is an integer, for example, fromabout 1 to about 500 (e.g., 1, 3, 5, 10, 20, 50, 100, 200, 500).

In certain embodiments, the molar ratio of

is from about 1:0.1 to about 0.1:1 (e.g., from about 0.05:1 to about1:0.05, from about 0.1:1 to about 1:0.1, from about 0.5:1 to about1:0.5, from about 0.8:1 to about 1:0.8).

The polymer and co-polymer of the invention may have a Mn from about5,000 to about 100,000 (e.g., 5k, 10k, 20k, 50k, 100k).

The invention also encompasses any product that is comprised of apolymer or co-polymer disclosed herein including any product preparedfrom a polymer or co-polymer of disclosed herein such as bypost-polymerization functionalization.

In yet another aspect, the invention generally relates to athree-dimensional scaffold comprising a polymer or co-polymer of theinvention and optionally includes cells, for example, selected fromosteoblasts, chondrocytes, endothelial cells, epithelial cells,embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells,or cell lines.

The polymer and co-polymer of the invention may be polymerized using asmall molecule initiator that includes an —OH, —SH or —NH₂ group, forexample, R_(sm)—OH, R_(sm)—SH or R_(sm)—NH₂, wherein R is anunsubstituted or substituted alkyl group or aryl group and one or morecarbon atoms of the alkyl or aryl group may be substituted with a heteroatom such as O, S or N. Non-limiting exemplary small molecule initiatorsinclude ethanol, benzyl alcohol, and ethylene glycol.

The polymer and co-polymer of the invention may be polymerized using apre-polymer initiator that includes an —OH, —SH or —NH₂ group, forexample, R_(pp)—OH, R_(pp)—SH or R_(pp)—NH₂, wherein R_(pp) is anoligomeric or polymeric group.

The polymerization initiator, either a small molecule or a pre-polymer,may include two or a multiple of —OH, —SH or NH₂ groups. For example, amulti-functional initiator may generate polymerization products havingbranched, star- or web-shaped polymer products,

In another aspect, the invention generally relates to a product thatincludes a polymer or a co-polymer of the invention.

The invention offers a number of distinct advantages. First, monomerpreparation and purification are simple. Compared with existingfunctional monomers which usually involve multiple reaction steps withchromatographic purifications and low yields, the monomers of theinvention can be prepared in good overall yield (e.g., about 50% orgreater) with high purity (e.g., >99%) in just two steps, without theneed for any chromatography.

Additionally, monomers of the invention are compatible with existingliving polymerization techniques and may be polymerization in bothmelting and solution state catalyzed by metal catalysts or all-organiccatalysts. The azido groups do not interfere with the polymerizationstep and are compatible with most existing ring-opening livingpolymerization catalytic systems. The monomers of the invention are alsocompatible with copolymerization with industrial monomers. For example,the monomers may be copolymerized with L-lactide, D,L-lactide,e-caprolactone, and glycolide, which are commonly used in industrial ROPprocesses.

Furthermore, homopolymers and copolymers of the invention arewell-defined products, e.g., with high molecular weight (>10,000) andnarrow polydispersity (PDI<1.1). Linear, branched and star architecturewith random, block or multi-segment compositions may be prepared bychoosing suitable initiator, co-monomer and monomer feeding ratios andsequences. For example, the azido-containing homopolymers and copolymersmay be prepared that are stable up to 175° C., making them compatiblewith industrial thermal processing techniques.

Another distinctive advantage is that post-polymerizationfunctionalization can be performed under mild conditions. For instance,the appended azido groups along the polymer backbone can be directlymodified with small molecule, oligomers and biomacromolecules throughcopper-catalyzed or copper-free “click” chemistries with high fidelityunder mild conditions. Degradation of polymers can be prevented undersuch mild conditions, especially those strain-promoted, copper-free“click” reactions that eliminate the use of toxic copper catalysts. Suchan approach may be particularly attractive for making materials for invivo applications.

Cytocompatible Poly(Ethylene Glycol)-Co-Polycarbonate Hydrogels

The invention also provides novel and efficient methods for preparingcytocompatible poly(ethylene glycol)-co-polycarbonate hydrogels, forexample, crosslinked by copper-free, strain-promoted “click” chemistry.The invention discloses a facile method for preparing PEG macromersflanked with aliphatic azido-functionalized biodegradable polycarbonateblocks, which are subsequently crosslinked with dibenzylcocloctyne(DBCO)-terminated PEG macromers using copper-free, strain-promoted [3+2]azide-alkyne cylcloaddition (SPAAC). The choice of the SPAAC “click”chemistry as the in situ crosslinking strategy is to take advantage ofthe high fidelity and orthogonality of the reaction as well as itscompatibility with physiological conditions. (Agard, et al. J. Am. Chem.Soc. 2005, 127, 11196; Baskin, et al. Aldrichimica Acta 2010, 43, 15.)Equally important, comparing to the copper-catalyzed [3+2] azide-alkynecylcloaddition (CuAAC) that have been more broadly applied to thefunctionalization of hydrogels including PEG, PVA and hyaluronan, thecopper-free SPAAC presents significant advantage in terms of bothshort-term cytocompatibility and long-term biocompatibility. (Malkoch,et al. Chem. Commun. 2006, 2774; Ossipov, et al. Macromolecules 2006,39, 1709; Crescenzi, et al. Biomacromolecules 2007, 8, 1844.)

Although more biocompatible metal catalysts are being developed forCuAAC, their safety for in vivo tissue engineering applications remainsunknown. By contrast, the SPAAC has already been utilized for live cellimaging, in vivo metabolic labelling in C. elegans, zebrafish and mice.(Amo, et al. J. Am. Chem. Soc. 2010, 132, 16893; Beatty, et al. Chem BioChem 2010, 11, 2092; Laughlin, et al. ACS Chem. Biol. 2009, 4, 1068;Laughlin, et al. Science 2008, 320, 664; Baskin, et al. Proc. Natl.Acad. Sci. U.S.A. 2010, 107, 10360; Chang, et al. Proc. Natl. Acad. Sci.U.S.A. 2010, 107, 1821.) Thus, it is not surprising that SPAAC hasquickly caught the attention of the polymer/hydrogel and tissueengineering communities. (DeForest, et al. Chem. Mater. 2010, 22, 4783;DeForest, et al. Nat. Mater. 2009, 8, 659.)

The azido-functionalized polycarbonate blocks, which serve as SPAACcrosslinking sites, are grafted to the ends of the hydrophilic PEG usingorganocatalytic ring-opening polymerization (ROP) of an easy-to-preparefunctional cyclic carbonate monomer, AzDXO, that was recently developed.(Xu, et al. Macromolecules, 44, 2660.) ROP of cyclic monomers has beenused for preparing biodegradable polymers. Recent progress on thedevelopment of organocatalysts for ROP offers great potential forpreparing hydrogels with reduced toxicity that are associated withtraditional metal catalysts. (Kamber, et al. Chem. Rev. 2007, 107, 5813;Nederberg, et al. Soft Matter 2010, 6, 2006; Nederberg, et al.Biomacromolecules 2007, 8, 3294.) The previously demonstrated(co)polymerization versatility of AzDXO by living organocatalytic ROPunder mild conditions (e.g., rt) and the facile functionalization of theside chains of the resulting polycarbonate P(AzDXO) via SPAAC underphysiological conditions have opened the possibilities for adjusting themechanical, biochemical and degradation properties of thePEG-co-P(AzDXO) hydrogels for regenerative medicine applications. (Xu,et al. Macromolecules, 44, 2660.)

Cytocompatible PEG-co-polycarobonate hydrogels crosslinked by watersoluble PEG-P(AzDXO)_(2m) macromers with varying PEG block lengths andlinear or 4-armed DBCO-capped PEG macromers were prepared usingcopper-free SPAAC. The macromer components were non-cytotoxic, and therapid gelling (as quick as <60 sec) enabled by the copper-free “click”chemistry allowed the encapsulation of bone-marrow derived stromal cellswith higher cellular viability than the photo-crosslinked PEG6k-DMA gelscommonly used for cartilage tissue engineering applications. Themechanical properties of these gels could be readily tuned by theadjusting macromer structures and the lengths of their constituentpolymer blocks. The combination of cytocompatibility and tunable gellingrates and mechanical properties make these “clickable” gels appealingcandidates as cell encapsulation strategies and as injectableformulations for minimally invasive tissue repair.

Thus, in yet another aspect, the invention generally relates to a methodfor preparing a hydrogel of poly(ethylene glycol)-co-polycarbonate. Themethod includes: (1) providing a first poly(ethylene glycol) macromercomprising a poly(ethylene glycol) oligomer segment and a flankingsegment on one or more side thereof, wherein each flanking segmentcomprises a single- or multi-azido-functionalized biodegradablepolycarbonate block; (2) providing a second poly(ethylene glycol)macromer comprising a poly(ethylene glycol) oligomer segment and aflanking segment on one or more side thereof, wherein each flankingsegment comprises a terminal alkyne group; and (3) forming a hydrogel bycrosslinking the first macromer and the second macromer under conditionsso as to effect azide-alkyne cycloaddition.

In certain preferred embodiments, the method further includes: providingcells prior to the crosslinking step; and crosslinking the firstmacromer and the second macromer in the presence of cells underconditions so as to effect copper-free, strain-promoted azide-alkynecycloaddition.

In some embodiments, the first poly(ethylene glycol) macromer and one ormore of the flanking segments thereof independently includes apoly(carbonate) block. In some embodiments, the second poly(ethyleneglycol) macromer and one or more of the flanking segments thereofindependently comprises a poly(carbonate) block.

In certain preferred embodiments, the first poly(ethylene glycol)macromer has the structural formula of:

wherein each R is a group that comprises either an azide group or analkynyl group, and n is an integer from about 0 to about 5,000 (e.g., 0,1, 5, 10, 20, 50, 100, 500, 1,000, 2,000, 5,000). For example, each Rmay independently be

wherein each of R₃ and R₄ independently in hydrogen, an alkyl, analkoxy, —OH, a halide, an aryl, an aryloxy, an acyl, a haloalkyl group,provided that one or both of R₃ and R₄ independently is independentlyR_(x), wherein R_(x) is a group that comprises either an azide group oran alkynyl group, and n is an integer from about 1 to about 2,000 (e.g.,1, 5, 10, 20, 50, 100, 200, 500, 1000, 2000).

In certain embodiments, the second poly(ethylene glycol) macromer hasthe general structural formula of:

wherein R is

and each of a, b, c, and d is an integer and from about 1 to about 2000(e.g., 1, 5, 10, 20, 50, 100, 200, 500, 1000, 2000).

In some embodiments, each of the flanking segment of the firstpoly(ethylene glycol) macromer comprises a multi-azido-functionalizedbiodegradable polycarbonate block. In some embodiments, each of theflanking segment of the second poly(ethylene glycol) macromer comprisesan acyclic terminal alkyne group.

In some embodiments, each of the flanking segment of the secondpoly(ethylene glycol) macromer comprises a cyclic terminal alkyne group,for example, a dibenzylcyclooctyne (DBCO) group.

In certain preferred embodiments, crosslinking the first macromer andthe second macromer is performed via copper-free, strain-promotedazide-alkyne cycloaddition. In some embodiments, crosslinking the firstmacromer and the second macromer is performed via copper-catalyzedazide-alkyne cycloaddition.

Any suitable biological cells may be encapsulated via methods disclosedherein, for example, mammalian cells including osteoblasts,chondrocytes, endothelial cells, epithelial cells, embryonic stem cells,mesenchymal stem cells, hematopoietic stem cells, or cell lines. Incertain preferred embodiments, the encapsulated cells are bone marrowstromal cells or mesenchymal stem cells.

In yet another aspect, the invention generally relates to a hydrogelcomposition that includes a hydrogel composition comprising acrosslinked product of a first macromer and a second macromere. Thefirst macromer comprises a water-soluble hydrophilic polymer segment anda flanking segment on one or more side thereof, wherein each flankingsegment comprises a single- or multi-azido-functionalized biodegradablepolycarbonate block. The second macromer comprises a water-solublehydrophilic polymer segment and a flanking segment on one or more sidethereof, wherein each flanking segment comprises a terminal alkynegroup.

In certain preferred embodiments, the first macromer is a firstpoly(ethylene glycol) macromer and the second macromer is a secondpoly(ethylene glycol) macromere. The first poly(ethylene glycol)macromer comprises a poly(ethylene glycol) oligomer segment and aflanking segment on one or more side thereof, wherein each flankingsegment comprises a single- or multi-azido-functionalized biodegradablepolycarbonate block. The second poly(ethylene glycol) macromer comprisesa poly(ethylene glycol) oligomer segment and a flanking segment on oneor more side thereof, wherein each flanking segment comprises a terminalalkyne group.

In yet another aspect, the invention generally relates to acytocompatible hydrogel composition suitable for use in tissue repair orregeneration, comprising a three-dimensional construct of cells and acrosslinked network of a first poly(ethylene glycol) macromer and asecond poly(ethylene glycol) macromere. The first poly(ethylene glycol)macromer comprises a poly(ethylene glycol) oligomer segment and aflanking segment on one or more side thereof, wherein each flankingsegment comprises a single- or multi-azido-functionalized biodegradablepolycarbonate block. The second poly(ethylene glycol) macromer comprisesa poly(ethylene glycol) oligomer segment and a flanking segment on oneor more side thereof, wherein each flanking segment comprises a terminalalkyne group.

The invention also encompasses any product that is comprised of ahydrogel (with or without encapsulated cells) disclosed herein includingany product prepared from a hydrogel disclosed herein such as bypost-polymerization functionalization.

Examples Monomers and Polymers for Well-Defined FunctionalPolycarbonates and Poly(Ester-Carbonates)

Monomer Design and Preparation.

High molecular weight polyesters and polycarbonates are usually preparedby ring-opening polymerization (ROP) of cyclic monomers. (Dechy-Cabaret,et al. Chem. Rev. 2004, 104, 6147.) Conventional catalysts andpolymerization conditions employed in ROP could result in relativelybroad molecular weight distributions and unexpected ether formations.(Rokicki, G. Prog. Polym. Sci. 2000, 25, 259; Ariga, et al.Macromolecules 1997, 30, 737.) Recent developments on organic catalystsfor ROP have made it possible to prepare well-defined polyesters andpolycarbonates (PDI<1.1) under mild conditions. (Nederberg, et al.Biomacromolecules 2007, 8, 153; Kamber, et al. Chem. Rev. 2007, 107,5813.) A general method for preparing functional poly(ester-carbonates)with well-defined compositions and structures, however, is yet to bedeveloped. Our goal is to develop an azido-functionalized cycliccarbonate monomer that exhibits ROP kinetics similar to that of the ROPof lactides under the same mild conditions enabled by organic catalyst1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU). Such an approximation ofpolymerization kinetics could enable poly(ester-carbonates) with bothrandom and block architectures be prepared with well-definedcompositions and narrow polydispersity. The choice of the azidofunctionality over alkyne or acrylate as a “clickable” group of themonomer is mainly inspired by its ability to withstand most basic andacidic conditions as well as temperatures up to 130° C. where alkynesand acrylates tended to self-crosslink or participate in free radicalpolymerizations. (Brase, et al. Angew. Chem., Int. Ed. 2005, 44, 5188;Kuhling, et al. Macromolecules 1990, 23, 4192; Sumerlin, et al.Macromolecules 2005, 38, 7540.) In addition, the availability of manycommercialized alkyne and cyclooctyne derivatives makes thepost-polymerization functionalization with or without copper catalyststraightforward. Finally, the azido groups could also enablefunctionalizations via Staudinger ligation, which could be useful forcertain biological investigations both in vitro and in vivo. (Baskin, etal. Aldrichimica Acta 2010, 43, 15; Saxon, et al. Science 2000, 287,2007.)

For example, 5,5-bis(azidomethyl)-1,3-dioxan-2-one (AzDXO, FIG. 1a ) wasdesigned as the functional monomer. AzDXO was synthesized from2,2-bis(bromomethyl)propane-1,3-diol in 2 steps with an overall yield of45.6%. High purity (>99%) product was obtained by recrystallizationpurification. Despite its high azido content, the monomer was safe tohandle during room temperature. No decomposition or explosion wasobserved when it was vacuum-dried at 90° C. for 2 days, although likeall azido compounds, this monomer should always be handled withprecaution.

FIG. 1 shows the synthesis and polymerization kinetics ofazido-functionalized cyclic carbonate monomer AzDXO. (a) AzDXO wassynthesized in 2 steps in 45.6% overall yield: i) NaN₃, DMSO, 110° C.,16 h; ii) ethyl chloroformate, THF, ice bath 4 h, rt (room temperature)12 h. Ring opening polymerization of AzDXO was carried out in CDCl₃ atrt using benzyl alcohol (Bz) as an initiator and1,8-Diazabicyclo[5.4.0]-undec-7-ene (DBU) as a catalyst. [AzDXO]₀=1.0 M;[AzDXO]₀: [Bz]: [DBU]=50:1:0.01. (b) GPC monitoring of polymerization byevaporative light scattering (ELS) detector over time; (c)Polymerization kinetics determined from GPC showing linear increase ofnumber-average molecular weight (M_(n)) and consistent lowpolydispersity (PDI) with the increase of monomer conversion,respectively. (d) Plot of ln([M]₀/[M]) vs time (t) showing a linearfirst-order kinetics for the ROP of AzDXO, supporting acontrolled/“living” polymerization mechanism. [M]₀ and [M] are themonomer concentrations at time zero and time t, respectively, and t isthe reaction time in minutes.

Polymerization Kinetics.

ROP of AzDXO was carried out at room temperature in chloroform with DBU(0.01M) as the catalyst. The polymerization kinetics was studied usingbenzyl alcohol (Bz) as an initiator (FIG. 1a ) and monitored by gelpermeation chromatography (GPC) and ¹H NMR spectroscopy. Thepolymerization occurred instantly upon the addition of DBU, as supportedby the decrease of the intensity of the GPC signal of the monomer (at19.35 min) and the appearance of a new, earlier-eluting peak (around18.10 min) as early as at 20 sec (FIG. 1b ). The polymerization wasalmost completed by 60 min as supported by the disappearance of themonomer peak. ¹H NMR monitoring (FIG. 7) revealed similar polymerizationkinetics. The number-averaged molecular weight (M_(n)) ofBz-P(AzDXO_(n)) determined from GPC exhibited a linear positivecorrelation with the monomer conversion, with the low polydispersityindex (PDI<1.1) maintained as the M_(r), increased (FIG. 1c ),supporting that the initiating sites remained active during thepolymerization. The minimal increase of PDI at very high conversion (PDIstill well below 1.1) was likely due to decreased solubility of thehigher molecular weight active species and led to some chaintermination. It was previously shown that under the catalysis of DBU,cyclic carbonate and lactone could be polymerized in controlled/“living”anionic ROP, with a presumed hydrogen-bonding interaction between theinitiating alcohol and the nucleophilic nitrogen of DBU. (Endo, et al.Macromolecules 2005, 38, 8177; Kiesewetter, et al. Macromolecules 2010,43, 2093.) Our results indicate that the ROP of AzDXO proceeded in asimilar fashion. The plot of ln([M]₀/[M]) versus time revealed a linearfirst-order polymerization kinetics (FIG. 1d ), indicating that theconcentration of the growing end remained constant during polymerizationand further supporting the living polymerization mechanism. The ROP rateconstants of AzDXO and L-lactide (L-LA) under identical reactionconditions were determined as 0.084 min⁻¹ and 0.116 min⁻¹, respectively.The similar rate constants make it possible to prepare copolymers ofL-LA and AzDXO in a controlled manner without further changing thepolymerization conditions.

Thermal Properties of Homopolymers.

The thermal properties of homopolymer Bz-P(AzDXO_(n)) (n=20, 40 or 80)were analyzed by differential scanning calorimetry (DSC). Allhomopolymers exhibited large endothermic melting peaks around 50-100° C.(FIG. 2), with melting point (T_(m)) increased from 86.31 to 98.43° C.while melting enthalpy (AH) remained constant around −52.0 J/g as thechain length increased from 20 to 80 repeating units (Table 1). Afterquenching the melts to −50° C., a glass transition slightly below 0° C.was detected from the second heating scan in all homopolymers, with theT_(g) slightly increasing as the chain length increased. In comparison,the most studied aliphatic polycarbonate poly(trimethylene carbonate) ofcomparable molecular weights exhibited T_(g)'s ranging from −26 to −15°C., a T_(m) of 36° C. and ΔH_(m)'s of −4.5 to 10 J/g. (Zhu, et al.Macromolecules 1991, 24, 1736.) FIG. 2 shows differential scanningcalorimetry of homopolymers of AzDXO with different molecular weights.The solid and dotted curves denote the first and the second DSC cycles,respectively.

TABLE 1 Homopolymers of AzDXO and their thermal properties Name AzDXO:BAM_(n) ^(a) PDI^(a) T_(m) (° C.)^(b) ΔH (J/g)^(b) T_(g) (° C.)^(c) Bz-20:1 5245 1.1512 86.31 −52.49 −5.1 P(AzDXO)₂₀ Bz- 40:1 8546 1.0833 95.21−53.16 −2.4 P(AzDXO)₄₀ Bz- 80:1 14886 1.0626 98.43 −51.93 −1.7P(AzDXO)₈₀ ^(a)Measured by GPC (THF, 0.3 mL/min, rt), calibrated bypolystyrene standards ^(b)Determined from the endothermic peak maximumin the first heating cycle of the DSC scans ^(c)Determined from themidpoint of the first endothermic transition in the second heating cycleof the DSC scans

Copolymerization of AzDXO and L-LA.

The comparable DBU-catalyzed homopolymerization rates observed withAzDXO and L-LA make the preparation of copolymers of AzDXO and L-LAstraightforward. Using ethylene glycol as an initiator, randomcopolymers with varying compositions and narrow PDI (˜1.1, Table 2) weresuccessfully prepared by simultaneous addition of the two monomers (FIG.3).

By changing the monomer feed ratios during the one-pot randomcopolymerization, materials with altered crystallinity and thermalproperties could be prepared. The T_(m)'s and ΔH_(m)'s of the randomcopolymers decreased with the increase of AzDXO content (FIG. 8, Table2) while their physical appearance changed from crystalline whitepowders to semi-crystalline translucent solids to amorphous transparentresins. Although homopolymers EG-P(LLA₁₀₀) and EG-P(AzDXO₁₀₀) are bothhighly crystalline materials with a AH of −50.0 J/g and −47.8 J/g,respectively, their equal molar ratio random copolymerEG-P(LLA₅₀-co-AzDXO₅₀) was amorphous as indicated by the lack of meltingpeaks (FIG. 8d ), supporting a random distribution of AzDXO and L-LAalong the copolymer chains.

TABLE 2 Random Copolymers of AzDXO with L-Lactide and their thermalproperties Name L-LA:AzDXO:EG L-LA:AzDXO^(a) M_(n) ^(b) PDI^(b) T_(m) (°C.)^(c) ΔH (J/g)^(c) T_(g) (° C.)^(d) EG-P(LLA₁₀₀) 100:0:1 N.A. 212991.11 145.5 −47.8 47.2 EG-P(LLA₉₅-co- 95:5:1 95:5.0 18727 1.12 137.3−35.3 48.1 AzDXO₅) EG-P(LLA₉₀-co- 90:10:1 90:9.8 22480 1.10 130.9 −28.445.0 AzDXO₁₀) EG-P(LLA₈₀-co- 80:20:1 80:20.8 22652 1.11 107.8 −17.1 37.1AzDXO₂₀) EG-P(LLA₅₀-co- 50:50:1 50:52.5 21157 1.11 N.A. 0 18.2 AzDXO₅₀)^(a)Based on the ¹H NMR integrations assuming that the L-lactide unitswere incorporated at theoretical values. ^(b)Measured by GPC (THF, 0.3mL/min, rt), calibrated by polystyrene standards. ^(c)Determined fromthe endothermic peak maximum in the first heating cycle of the DSCscans. ^(d)Determined from the midpoint of the first endothermictransition in the second heating cycle of the DSC scans.

Block copolymers tethered by two or more distinct chain segments canexhibit unique properties distinctive from those of the correspondinghomopolymers and random copolymers. (Hadjichristidis, et al. Blockcopolymers: synthetic strategies, physical properties, and applications;John Wiley and Sons, 2002.) Block copolymers can be prepared bysequentially growing the blocks using different polymerizationtechniques following proper end-group manipulations. (Hedrick, et al.Macromolecules 1998, 31, 8691; Feng, et al. Macromolecules 2002, 35,2084; Hawker, et al. Macromolecules 1998, 31, 213; Matyjaszewski, K.Macromol. Symp. 1998, 132, 85.) Alternatively, they can be preparedusing the same polymerization technique (e.g. Atom Transfer RadicalPolymerization and Reversible Addition-Fragmentation Chain TransferPolymerization) under identical reaction conditions by feeding monomersof interest in batches. (Matyjaszewski, et al. Chem. Rev. 2001, 101,2921; Chong, et al. Macromolecules 1999, 32, 2071.) The latter strategyeliminates the need for end group functionalization and is potentiallyfar more efficient during scale-ups. The incompatibility of many organicfunctional groups with ROP conditions, however, has made it difficult tobroadly apply such a strategy to the preparation of functionalbiodegradable block copolymers. Compatible with ROP conditions and witha ROP rate constant comparable to that of L-LA, AzDXO offers a uniqueopportunity to the efficient preparation of functionalpoly(ester-carbonate) block copolymers.

Block copolymer Pyrene-P(LLA_(x))-b-P(AzDXO_(y)) was prepared with fixedL-LA block length (x=100) and varying AzDXO block lengths (y=5, 10, 20,35) by DBU-catalyzed ROP at room temperature using 1-pyrenebutanol asthe initiator. 1-Pyrenebutanol was chosen for easy monitoring of thepolymerization by GPC and NMR due to its strong UV-vis absorption anddistinct down-field ¹H NMR signals away from those of the polymericunits. The rapid polymerization (<1 h) and high conversion of monomers(>99%) under the employed conditions enabled the sequential feed ofmonomers without the need for purification of the first polymer block.All block copolymers were prepared in quantitative yield with narrowpolydispersity (Table 3).

TABLE 3 Block and random copolymers of L-LA and AzDXO Name^(a)L-LA:AzDXO^(b) M_(n) ^(c) PDI^(c) Pyrene-P(LLA₁₀₀) 97.0:0   19392 1.06Pyrene-P(LLA₁₀₀)-b- 116.6:3.2  22356 1.06 P(AzDXO₅) Pyrene-P(LLA₁₀₀)-b-106.8:9.2  23487 1.06 P(AzDXO₁₀) Pyrene-P(LLA₁₀₀)-b- 110.8:18.3  273621.11 P(AzDXO₂₀) Pyrene-P(LLA₁₀₀)-b- 98.8:33.1 31030 1.07 P(AzDXO35)Pyrene-P(AzDXO₁₀₀)    0:106.0 19888 1.14 Pyrene-P(LLA₁₀₀-co- 98.0:18.625524 1.07 AzDXO₂₀)^(d) ^(a)The naming of the samples reflect thetheoretical copolymer compositions; ^(b)copolymer compositionsdetermined from ¹H NMR; ^(c)number-averaged molecular weight andpolydispersity index determined from GPC using an ELS detector; ^(d)twomonomers were added in one-step for the preparation of random copolymer.

Representative GPC traces for Pyrene-P(LLA₁₀₀) andPyrene-P(LLA₁₀₀)-b-P(AzDXO₃₅) (FIG. 4a ) show that both ELS and UV-vispeaks shifted to an earlier elution upon feeding the second monomerAzDXO while the peak shapes remained unchanged. The drop in intensity ofUV-vis signal upon the polymerization of the P(AzDXO₃₅) block supportsthat the polymerization of AzDXO was initiated by the Pyrene-P(LLA₁₀₀)block. ¹H and ¹³C NMR (FIGS. 9 and 10) showed two distinct groups ofpeaks correlating to the two functional blocks in the block copolymerwhereas broader and more scattered peaks for the random copolymer. Themicrostructural difference of the copolymers also translated intodistinctive thermal properties. As the AzDXO block grew longer after theL-LA block, the appearance of two distinct melting peaks and twodistinct glass transitions in the block copolymer became apparent (FIG.11). For instance, Pyrene-P(LLA₁₀₀)-b-P(AzDXO₂₀) exhibited two meltingpeaks (T_(m) ¹=80.6° C.; T_(m) ²=143.6° C.) and two glass transitions(T_(g) ¹=−1.5° C.; T_(g) ²=48.7° C.) that resembled the respectivetransitions for the homopolymers Pyrene-P(LLA₁₀₀) and P(AzDXO₁₀₀) (FIG.4b ), supporting the block microstructure. By contrast, the randomcopolymer Pyrene-P(LLA₁₀₀-co-AzDXO₂₀) only exhibited one small broadmelting peak around 117.8° C. and a single glass transition around 39.2°C., consistent with its more randomly distributed compositions and lessordered microstructure.

FIG. 4 shows GPC traces and DSC scans confirming the successfulpreparation of block and random copolymers of L-LA and AzDXO. (a)Representative GPC traces for Pyrene-P(LLA₁₀₀)-b-P(AzDXO₃₅) before andafter feeding AzDXO; (b) DSC curves of homopolymers, and block andrandom copolymers of L-LA and AzDXO.

Functionalization of Block Copolymers Via Copper-Catalyzed Azido-AlkyneCycloaddition (CuAAC).

To demonstrate the facile post-polymerization conversions of the azidofunctionality, copper-catalyzed azido-alkyne cycloaddition (CuAAC)reaction was carried out at room temperature in DMF to attach5-hexyn-1-ol (HX) to the block and random copolymers (FIG. 5a ). (Kolb,et al. Angew. Chem., Int. Ed. 2001, 40, 2004; Hawker, et al. Aust. J.Chem. 2007, 60, 381.) Nearly all azido groups were converted by 24 h, assupported by FTIR (FIG. 12) and 1H NMR spectra (FIG. 13). The GPCtraces, however, revealed complex elution profiles for HX-modified blockand random copolymers (FIG. 5b &c). The apparent M_(n) calculated fromthe main peaks showed lower value than the original polymers, indicatingthe decrease of hydrodynamic radii of the polymers upon CuAAC, likely asa result of the collapse of the hydrophilic HX side chains due to eitherpoor solvation or complexation with residue Cu²⁺ (Jiang, et al.Macromolecules 2008, 41, 1937.) The extra shoulder peaks at earlierelution time may have derived from copolymer aggregates formed via thetriazole-Cu² complexation. After intensive dialysis against Cu²⁺sequestrant 2,2′-bipyridine, these shoulder peaks was reduced althoughnever completely removed.

FIG. 5 shows functionalization of block and random copolymers with5-hexyn-1-ol (HX) via copper-catalyzed azido-alkyne cycloaddition(CuAAC). (a) Reaction scheme; (b) GPC traces (DMF, 50° C.), M_(n) andPDI of block copolymer P(LLA₁₀₀)-b-P(AzDXO₂₀) before and after CuAAC;(c) GPC traces (DMF, 50° C.), M_(n) and PDI of random copolymerP(LLA₁₀₀)-co-P(AzDXO₂₀) before and after CuAAC.

Functionalization of Block Copolymers Via Strain-Promoted Azido-AlkyneCyclcoaddition (SPAAC).

To avoid the potential toxicity of residual copper catalyst in CuAAC,copper-free, strain-promoted azido-alkyne cyclcoaddition (SPAAC) wascarried out to modify the block and random copolymers (FIG. 6a ) usingaza-dibenzocyclooctyne NHS ester (DBCO-NHS). (Baskin, et al.Aldrichimica Acta 2010, 43, 15; Agard, et al. J. Am. Chem. Soc. 2005,127, 11196.) Near complete conversion (>95%) of azido groups wasachieved in 4 h when 1 eq. DBCO-NHS was added in DMF. Completeconversion was achieved when the DBCO-NHS to azide ratio was increasedby 3 folds. GPC traces revealed the expected increase of molecularweight upon the modification of either block (FIG. 6b ) or randomcopolymer (FIG. 6c ) with the narrow polydispersity retained. ¹H NMR(FIG. 6d ) confirmed the complete disappearance of proton resonance for—CH₂N₃ at 3.50 ppm (FIG. 9) and the appearance of proton resonancesassociated with the covalently attached DBCO-NHS. The resonance around4.40 ppm corresponding to the AzDXO unit was more readily detected inPyrene-P(LLA₁₀₀-co-AzDXO₂₀)-DN than in Pyrene-P(LLA₁₀₀-b-AzDXO₂₀)-DN,supporting the random distribution of the bulky DN rings andconsequently the less profound shielding effect in the former.Consistent with this observation, higher peak intensities were alsodetected for the resonances corresponding to the LLA protons in therandom copolymers.

FIG. 6 shows GPC traces and ¹H NMR spectra indicated the successfulmodification of block and random copolymers by strain-promotedazido-alkyne cycloaddition (SPAAC). (a) reaction scheme; (b) GPC traces(ELS detector) of block copolymer Pyrene-P(LLA₁₀₀)-b-P(AzDXO₂₀) beforeand after SPAAC; (c) GPC traces (ELS detector) of random copolymerPyrene-P(LLA₁₀₀-co-AzDXO₂₀) before and after SPAAC; (d) ¹H NMR ofDBCO-NHS (DN) (top), Pyrene-P(LLA₁₀₀)-b-P(AzDXO₂₀)-DN (middle) andPyrene-P(LLA₁₀₀-co-AzDXO₂₀)-DN (bottom).

Experimental Section

Materials and Instrumentation:

Aza-dibenzocyclooctyne NHS ester (DBCO-NHS) was purchased from ClickChemistry Tools Inc. (Macon, Ga., USA). All other chemicals werepurchased from Sigma-Aldrich (St. Louis, Mo.) and used as receivedunless otherwise specified. Chloroform and deuterated chloroform usedfor reactions were pre-dried by refluxing with phosphorus pentoxideovernight and then distilled under argon. Triethyl amine (TEA, >99%) wasdried by refluxing with calcium hydride and distilled under argon.(3S)-Cis-3,6-dimethyl-1,4-dioxane-2,5-dione (L-lactide, 98%) was furtherpurified prior to use by repeated (3×) recrystallization from anhydroustoluene. 1,8-Diazabicyclo[5.4.0]-undec-7-ene (DBU, 98%) was freshlydistilled under vacuum prior to use.

¹H (400 MHz) and ¹³C NMR (100 MHz) spectra were recorded on a VarianINOVA-400 spectrometer in deuterated chloroform (CDCl₃, 99.8 atom % Dwith 0.03% v/v TMS) or dimethyl sulfoxide-d6 (99.9 atom % D with 0.03%v/v TMS). High resolution mass spectroscopy (HRMS) spectra were recordedon a Waters Q-T of Premier mass spectrometer using electro sprayionization (ESI) with W-mode and a spray voltage of 3500V. Samples (0.1or 10 mg/mL) in methanol or methanol/water mixture (50:50) were infusedat a rate of 5 μl/min. Fourier transformed infrared spectroscopy (FTIR)spectra were taken on a Thermo Electron Nicolet IR100 spectrometer with2-cm⁻¹ spectral resolution. Liquid samples were coated on a NaCl saltwindow and solid samples were mold-pressed into thin transparent discswith KBr, respectively.

Gel permeation chromatrography (GPC) measurements were taken on a VarianProstar HPLC system equipped with two 5-mm PLGel MiniMIX-D columns(Polymer Laboratory, Amherst, Mass.), a UV-vis detector and a PL-ELS2100evaporative light scattering detector (Polymer Laboratory, Amherst,Mass.). For characterization of azido-functionalized polycarbonates andpoly(ester-carbonates), THF was used as an eluent at a flow rate of 0.3mL/min at rt. The number-averaged molecular weight (M_(n)) and thepolydispersity index (PDI) were calculated by a Cirrus AIA GPC Softwareusing narrowly dispersed polystyrenes (ReadyCal kits, PSS PolymerStandards Service Inc. Germany) as calibration standards. Forcharacterization of the more polar functional polymers following “click”chemistry, DMF was used as an eluent at a flow rate of 0.3 ml/min at 50°C. The M_(n) and PDI were calculated by using narrowly dispersedpoly(methyl methacrylates) (EasiVial, Polymer Laboratory, Amherst,Mass.) as standards.

The thermal properties of polymers were determined on a TA InstrumentsQ200 Differential Scanning calorimeter (DSC). The enthalpy (cellconstant) and temperatures are calibrated by running a high-purityindium standard under the conditions identical to those used for samplemeasurements. To determine the crystallinity and glass transitions ofthe polymer samples, each specimen (around 5 mg) was subject to twoscanning cycles: (1) cooling from 40° C. (standby temperature) to −50°C. at −10° C./min, equilibrating for 2 min before being heated to 175°C. at a heating rate of 10° C./min; (2) cooling to −50° C. at −10°C./min, equilibrating for 2 min, and then being heated to 175° C. at arate of 10° C./min. The endothermic peak maximum in the first heatingcycle was recorded as the melting temperature (T_(m)), and itsassociated peak integration was calculated as melting enthalpy (ΔH). Themidpoint of the first endothermic transition from the second heatingcycle was identified as the glass transition temperature (T_(g)).

Monomer Synthesis and Polymerizations2,2-bis(azidomethyl)propane-1,3-diol

2,2-Bis(bromomethyl)propane-1,3-diol (98%, 104.7 g, 0.40 mol) wasdissolved in DMSO (300 mL) in a 1000-mL 3-neck flack equipped with areflux condenser, to which sodium azide (65.0 g, 1.0 mol) was addedunder argon. The suspension was heated to 110° C. and stirred for 16 h.After being cooled to rt, 150-mL water was added and the mixture wastransferred to a 2-L separatory funnel and extracted with 800-mL ethylacetate 3 times. The combined organic phase was washed by 200-mLsaturated brine 3 times and dried with sodium sulfate. Pale yellow oil(71.0 g, 95.3% yield) was obtained after removing the volatiles undervacuum. ¹H NMR (CDCl₃, 400 MHz, 20° C.): δ 3.629-3.616 (d, 4H, J=5.1Hz), 3.420 (s, 4H), 2.473-2.446 (t, 2H, J=5.1 Hz) ppm. ¹³C NMR (CDCl₃,100 MHz, 20° C.): δ 63.841, 51.907, 45.020 ppm. ESI-HRMS (m/z):C₅H₁₀N₆O₂Na⁺ [M+Na]⁺, calculated 209.0763. found 209.0754.

5,5-bis(azidomethyl)-1,3-dioxane-2-one (AzDXO)

To a solution of ethyl chloroformate (97%, 47.8 g, 0.44 mol) inanhydrous THF (300 mL) was added a THF solution (40 mL) of2,2-bis(azidomethyl)propane-1,3-diol (34.0 g, 0.18 mol) over 10 min at0° C. under argon. Dry TEA (50.0 g, 0.50 mol) was added slowly over 30min. The reaction was continued in ice bath for 4 h and then at rtovernight. After removing the white precipitate by filtration, thevolatile was removed under vacuum. The crude product was dissolved indichloromethane and passed through a short silica gel pad (230-400 meshsize) to remove salts. After removing the solvent by rotovapping, ethylether (250 mL) was added to dissolve the product and refluxed for 10min. Upon cooling, crystalline solid product was collected byfiltration. The recrystallization was repeated once. After dried undervacuum for 48 h, spectroscopically pure product (18.3 g) was obtained in47.9% yield. ¹H NMR (CDCl₃, 400 MHz, 20° C.): δ 4.251 (s, 4H), 3.550 (s,4H) ppm. 13C NMR (CDCl₃, 100 MHz, 20° C.): δ 147.33, 70.41, 50.34, 36.61ppm. ¹H NMR (DMSO-D6, 400 MHz, 40° C.): δ 3.59 (s, 4H) 4.27 (s, 4H) ppm;¹³C NMR (DMSO-D6, 100 MHz, 40° C.): δ 147.94, 70.90, 51.20, 36.87 ppm.ESI-HRMS (m/z): C₆H₈N₆O₃Na⁺ [M+Na]⁺, calculated 235.0556. found235.0556.

Typical Procedures for the Homopolymerization of AzDXO.

AzDXO (0.848 g, 4.0 mmol) and 800-μL benzyl alcohol solution (0.1M inchloroform) were mixed in a 6-mL reaction vessel under argon, to which3.0-mL chloroform was added to completely dissolve the monomer. Thepolymerization was initiated by the injection of 200-μL DBU solution(0.2 M in chloroform). The reaction was terminated after 2 h by addingbenzoic acid (99.5%, 12.2 mg, 0.1 mmol). The polymer was precipitated by50-mL methanol, redissolved in 4-mL chloroform and reprecipitated inmethanol. The precipitation purification was repeated twice to removeresidue catalyst and benzoic acid. The final precipitate was furtherwashed by ethyl ether and dried in vacuum oven at 60° C. for at least 48h prior to any thermal analysis. Around 0.820 g white powder productresulted (97% yield).

Typical Procedures for the Random Copolymerization of AzDXO with L-LA.

L-LA and AzDXO were polymerized in one-pot using CHCl₃ as solvent,ethylene glycol (EG) as the initiator, DBU (0.01M) as the catalyst, andwith the total monomer concentration kept at 1.0 M. The molar ratio ofAzDXO and L-LA was varied to prepare random block copolymers withdifferent AzDXO contents. For example, to preparedEG-F(LLA₉₀-co-AzDXO₁₀), chemicals at the molar ratio of[L-LA]₀:[AzDXO]₀:[EG]:[DBU]=90:10:1:0.01 were added. AzDXO (0.085 g, 0.4mmol), recrystallized L-LA (0.519 g, 3.6 mmol), and 400-μL ethyleneglycol solution (0.1 M in chloroform) were mixed in a 6-mL reactionvessel under argon, to which 3.4-mL chloroform was added to completelydissolve the monomer. The polymerization was initiated by the injectionof 200-μL DBU solution (0.2 M in chloroform). The reaction wasterminated after 2 h by adding benzoic acid, and the copolymerEG-P(LLA₉₀-co-AzDXO₁₀) was purified as described above and resulted in0.59 g white powder (98% yield).

Typical Procedures for the Sequential Copolymerization of AzDXO withL-LA.

L-LA and AzDXO were polymerized sequentially using CHCl₃ as solvent,1-pyrenbutanol as the initiator, DBU (0.01 M) as the catalyst, and with[L-LA]₀=1.0 M. The amount of AzDXO added next was varied to prepareblock copolymers with different AzDXO block lengths. For example, toprepared Pyrene-P(LLA₁₀₀)-b-P(AzDXO₂₀), chemicals at the molar ratio of[L-LA]₀: [AzDXO]₀:[1-pyrenebutanol]:[DBU]=100:20:1:0.01 were added inthe first step. Rrecrystallized L-LA (0.577 g, 4.0 mmol) and1-pyrenebutanol (99%, 10.9 mg, 0.040 mmol) were dissolved in 3.8-mLchloroform under argon. The polymerization was initiated by theinjection of 200-μL DBU solution (0.2 M in chloroform). After 2 h, AzDXO(0.169 g, 0.80 mmol) was added under argon and reacted for another 2 hbefore benzoic acid (≧99.5%, 12.2 mg, 0.1 mmol) was added to terminatethe polymerization. The product was purified as described above and thecopolymer Pyrene-P(LLA₁₀₀)-b-P(AzDXO₂₀) was obtained in the white powderform (0.72 g, 97% yield).

Side-Chain Functionalization by “Click” Chemistry:

Typical Procedures for the Functionalization of Poly(Ester-Carbonates)by Copper-Catalyzed Azido-Alkyne Cycloaddition (CuAAC).

The copolymers were functionalized in DMF at rt under inert atmospherewith varying amounts of 5-hexyn-1-ol by CuAAC. For example,Pyrene-P(LLA₁₀₀)-b-(AzDXO₂₀) or Pyrene-P(LLA₁₀₀-co-AzDXO₂₀) (30.0 mg,˜70 μmol of azido groups) and 5-hexyn-1-ol (96%, 120 mg, 1.23 mmol) weredissolved in 2.0 mL DMF, and degassed with argon for 30 min before CuBr(99.999%, 5 mg) was added. The reaction was allowed to continue at rtfor 24 h. The product was transferred to a 4-mL regenerated cellulosedialysis tubing (MWCO=3500 Dalton) and dialyzed against 400-mL DMF inthe presence of 0.1-g 2,2′-bipyridine to remove the copper catalyst. TheDMF solution was changed every two days for 7 days. After additionaldialysis against fresh DMF without 2,2′-bipyridine for another 2 days,the polymer solution in the dialysis tubing was dropped into 50-mL ethylether for precipitation. Light green powder (32.0 mg, yield≈88%) wasobtained after drying in vacuum oven at rt for 48 h.

Typical Procedures for the Functionalization of Poly(Ester-Carbonates)by Copper-Free Strain-Promoted Azido-Alkyne Cyclcoaddition (SPAAC).

The copolymers were functionalized in DMF at rt with varying amounts ofDBCO-NHS by SPAAC. For example, Pyrene-P(LLA₁₀₀)-b-P(AzDXO₂₀) orPyrene-P(LLA₁₀₀-co-AzDXO₂₀) (30.0 mg, ˜70 μmol of azido groups) andDBCO-NHS (101.0 mg, 210 μmol) were dissolved in 2.0-mL DMF, and reactedat rt for 12 h before being precipitated in 50-mL ethyl ether. Theprecipitate was redissovled in DMF and reprecipitated in ethyl ether.Off-white powder (˜60.0 mg, yield≈93%) was obtained after drying invacuum oven at rt for 48 h.

Synthesis of Mono Azido-Functional Cyclic Carbonate Monomer

Synthesis of 2-(bromomethyl)-2-methylpropane-1,3-diol (BMMPDiol)

Into a 500-mL 3-neck flask equipped with a condenser, a thermometric andargon inlet, 3-methyl-3-oxetanemethanol (20.4 g, 0.20 mol) was dissolvedin 250 mL THF and cooled in sodium chloride/ice bath. Aqueoushydrobromic acid (48 wt %, 80 mL) was dropped slowly in 1.5 h. Afterreacting in salt/ice bath for 4 h and then at room temperature foranother 12 h, 150 mL saturated sodium chloride solution was added. Thesolution was extracted with 250 mL of ethyl ether for 4 times. Thecombined ether solution was washed with 100 mL of saturated sodiumchloride solution twice and then dried over anhydrous sodium sulfate.The solution was filtered and the volatile was removed under vacuum,resulting in white powder (35.0 g, yield=96.1%). ¹H NMR (FIG. 14)(Chloroform-d, 400 MHz): δ(ppm) 3.68 (s, 4H), 3.56 (s, 2H), 0.93 (s,3H). ¹³C NMR (FIG. 15) (Chloroform-d, 100 MHz): δ (ppm) 68.2, 40.6,39.2, 18.4.

Synthesis of 2-(azidomethyl)-2-methylpropane-1,3-diol (AMMPDiol)

Into a 250-mL 3-neck flask equipped with a condenser, a thermometric andargon inlet, BMMPdiol (29.5 g, 0.16 mol) and sodium azide (26.0 g, 0.40mmol) was dissolved in 120 mL of dimethyl sulfone. After reacting at100° C. for 5 h, the solution was cooled down to room temperature and100 mL of water and 20 g of sodium chloride were added. The solution wasextracted with 200 mL ethyl ether for 4 times. The ether extract wascombined and washed with 100 mL of saturated sodium chloride solutiontwice. After dying over anhydrous sodium sulfate, the solution wasfiltered through a 2-cm silica gel pad (60-230 mesh). The volatile wasremoved under vacuum, resulting in colorless oil, which turned intowhite crystal over time (16.8 g, yield=72.3%). ¹H NMR (FIG. 16)(Chloroform-d, 400 MHz): δ(ppm) 3.59 (d, J=4.7 Hz, 4H), 3.44 (s, 2H),2.60-2.60 (t, J=4.7 Hz, 2H), 0.85 (s, 3H). ¹³C NMR (FIG. 17)(Chloroform-d, 100 MHz): δ (ppm) 68.0, 55.5, 40.9, 17.5.

Synthesis of 5-(azidomethyl)-5-methyl-1,3-dioxan-2-one

Into a 500-mL 3-neck flask equipped with a condenser, a thermometer andargon inlet, ethyl chloroformate (28.48 g, 0.262 mol) was added into 350mL of anhydrous tetrahydrofuran (THF) at −10° C. AMMPDiol (14.5 g, 0.10mol) was dropped in 10 min and then 43.0 mL of triethylamine was droppedslowly in 1 h. After reacting for 5 h in the salt/ice bath and 20 h atroom temperature, the solution was filtered and the filtrate wasconcentrated under vacuum. The resulting crude product was furtherpurified by recrystallization in ethyl ether, which was repeated twice.A white crystal was obtained (14.1 g, yield=82.4%). ¹H NMR (FIG. 18)(Chloroform-d, 400 MHz): δ(ppm) 4.18-4.28 (d, 2H), 4.05-4.16 (d, 2H),3.47 (s, 2H), 1.07 (s, 3H). ¹³C NMR (FIG. 19) (Chloroform-d, 100 MHz): δ(ppm) 147.6, 73.5, 53.8, 32.6, 17.2.

Synthesis of Hydrophilic Cyclic Carbonate Monomer with Poly(EthyleneGlycol) Side Chain.

A typical example of the synthesis of cyclic carbonate monomer with asingle poly(ethylene glycol) side chain (Mn=2000): Into a 100-mL 2-neckflask, methoxy poly(ethylene glycol) alkyne (Mn˜2000, 1.71 g, 0.857mmol) and AMMDXO (0.164 g, 0.958 mmol) were dissolved in 50 mL ofchloroform, and the solution was degassed and flushed with argon for 30min before copper wire (55 mg, 0.865 mmol) and copper bromide (18.4 mg,0.129 mmol) were added. After refluxing at 65° C. for 20 h, the solutionwas filtered and the filtrate was washed by 40 mL of 0.3M EDTA solution6 times. The solution was dried over anhydrous sodium sulfate and thenstirred with 4 g carbon black for 20 h. After filtering, the solutionwas concentrated to a final volume of ˜10 mL and dropped into 100 mL ofethyl ether, and the formed precipitate was collected by filtration.After drying under vacuum, 1.70 g white powder was obtained(yield=89.8%). ¹H NMR (FIG. 20) (Chloroform-d, 400 MHz): δ(ppm) 7.69 (s,1H), 4.72 (s, 2H), 4.48 (s, 2H), 4.13-4.29 (m, 4H), 3.42-3.88 (m, 194H),3.39 (s, 3H), 1.77 (s, 15H), 1.12 (s, 3H). ¹³C NMR (FIG. 21)(Chloroform-d, 100 MHz): δ (ppm) 147.2, 145.4, 124.4, 73.6, 71.7, 70.3,69.7, 69.0, 64.4, 58.8, 51.9, 33.1, 17.2.

Cytocompatible Poly(Ethylene Glycol)-Co-Polycarbonate Hydrogels

As disclosed herein, the invention additionally provides a facile methodfor preparing PEG macromers flanked with aliphatic azido-functionalizedbiodegradable polycarbonate blocks (FIG. 22a ), which are subsequentlycrosslinked with dibenzylcocloctyne (DBCO)-terminated PEG macromers(FIG. 22b ) using copper-free, strain-promoted [3+2] azide-alkynecylcloaddition (SPAAC) (FIG. 22c ).

The AzDXO monomer was synthesized in 2-steps as previously described.(Xu, et al. Macromolecules, 44, 2660.) Living/controlled ROP of AzDXO(0.1125 M) was initiated by varying amount of PEG6k, PEG10k and PEG20kusing organocatalyst DBU in dichloromethane (0.01 M) at rt (FIG. 22a ).The conversion of monomer reached −90% in 4 h. Upon termination bybenzoic acid, the PEG-P(AzDXO)_(2m) macromer products were purified byrepeated precipitation from dichloromethane in ethyl ether with >95%overall yield. As shown in Table 4, the P(AzDXO) block lengths (ordegree of polymerization, DP) in the resulting tri-block macromersPEG-P(AzDXO)_(2m) as determined by ¹H NMR (FIG. 23) were close to thetheoretical values (2m) calculated assuming 100% monomer conversion. GPCresults revealed very narrow polydispersity of all PEG-P(AzDXO)_(2m)(PDI: 1.02-1.09) with various PEG and P(AzDXO) block lengthcombinations. The water solubility of macromer PEG-P(AzDXO)_(2m) wasdependent on the overall length of the P(AzDXO) blocks (Table 4). Whenthe overall P(AzDXO) block length was shorter than 8 repeating units,the resulting PEG-P(AzDXO)_(2m) was soluble in water regardless of thelength of the PEG block.

By contrast, the triblock macromers became insoluble in water when thedegree of polymerization of AzDXO was above the critical number of 8regardless of the length of the initiating PEG (MW 6,000, 10,000 or20,000). Similar phenomenon was observed in other amphiphilic triblockcopolymers initiated by PEG. For example, Christine found that thetriblock PEG-(PLA)₂ copolymers were insoluble in water when the lacticacid repeating units were greater than 22, regardless of the PEG blocklength investigated. (Hiemstra, et al. Macromol. Symp. 2005, 224, 119.)

TABLE 4 Characterizations of PEG-P(AzDXO)_(2m) macromers Water Name^(a)DP^(b) M_(n) ^(NMR c) M_(n)/N₃ ^(d) M_(n) ^(GPC e) PDI^(e) solubilityPEG6k- 4.2 7909 946 12357 1.02 soluble P(AzDXO)₄ PEG6k- 6.8 8465 62212641 1.03 soluble P(AzDXO)₇ PEG6k- 8.4 8805 523 12931 1.03 cloudyP(AzDXO)₈ PEG6k- 10.8 9313 431 13168 1.03 insoluble P(AzDXO)₁₁ PEG10k-11.1 14658 662 20956 1.03 insoluble P(AzDXO)₁₁ PEG20k- 3.6 22338 307936563 1.08 soluble P(AzDXO)₄ PEG20k- 9.4 23573 1248 33380 1.09 swellableP(AzDXO)₉ ^(a)The naming of the samples reflects the approximatecopolymer compositions including the averaged molecular weight of theinitiating PEG and the degree of polymerization of the polycarbonateblocks; ^(b)degree of polymerization of AzDXO determined from ¹H NMR;^(c)number-averaged molecular weight calculated from ¹H NMR;^(d)number-averaged molecular weight per azido group;^(e)number-averaged molecular weight and polydispersity index determinedby GPC using an evaporative light scattering (ELS) detector.

The linear or 4-arm DBCO-terminated PEG macromers, PEG-(DBCO)_(x) (x=2or 4), were synthesized by end-capping PEGs of various molecular weightsand architectures (PEG6k, PEG20k or 4-arm-PEG10k) with DBCO-acid viaesterification. The reaction were carried out in anhydrousdichloromethane with catalysts N,N′-diisopropycarbodimide (DIPC) and4-(dimethylamino)-pyridinium p-toluenesulfonate (DPTS). The reactantratio was kept as 1:1.5:5:0.25/hydroxyl end-groups inPEG:DBCO-acid:DIPC:DPTS. Complete esterification of the PEG end-groupswas supported by the disappearance of the characteristic ¹³C NMR signalfor —CH₂—OH from 61.4 ppm in the ¹³C NMR spectra of PEG-(DBCO)_(x) (FIG.27). The catalysts and by-products were readily removed by washing withwater and subsequently precipitating in diethyl ether, or via sequentialdialysis precipitation against diethyl ether and water. An overall highyields of >90% were obtained. As representatively shown in FIG. 24, the¹H NMR peak at 2.17 ppm corresponding to the methylene protons of—CH₂COOH of DBCO-acid was shifted to 2.05 ppm upon esterification withPEG20k, and a new peak at 4.22 ppm corresponding to the methyleneprotons of —CH₂OCO— in the resulting PEG20k-(DBCO)₂ appeared. The peakat 6.07 ppm in PEG20k-(DBCO)₂ and the peak at 6.67 ppm in DBCO-acidcorresponded to the respective amide protons, the chemical shifts andintensities of which varied significantly with their concentrations andthe water content in the NMR solvent. The ¹³C NMR (FIG. 27) along withthe ¹H NMR integrations (FIG. 24) supported the successful attachment ofDBCO-acid to all hydroxyl termini of the PEG.

Water soluble PEG-P(AzDXO)_(2m) and PEG-(DBCO)_(x) were dissolved incell culture media, and in the presence of cell suspension, were readilymixed to form elastic cell-hydrogel construct under physiologicalconditions (FIG. 22d ). Six different hydrogels were prepared using twoPEG-P(AzDXO)_(2m) macromers and three PEG-(DBCO)_(x) macromers withdifferent structures, compositions, and molecular weights for thisstudy.

To study the SPAAC crosslinking process between the PEG-P(AzDXO)₂mmacromers and the PEG-(DBCO)_(x) macromers and to determine the shearmoduli of the crosslinked gels, time-sweep oscillatory rheology testswere carried out on the 6 formulations of the respective macromers (FIG.25). Briefly, PEG-(DBCO)2 or 4-arm-PEG-(DBCO)4 solution (10 w/v %) wasfirst loaded on the bottom plate of 20-mm parallel plates equipped witha Peltier heating unit (AR-2000 Rheometer, TA Instruments), before theaqueous solution of PEG-P(AzDXO)2m (10 w/v %) was added and rapidlymixed on plate by a pipette. The mixtures were equilibrated at 37° C.between the plates for 1 min prior to the test to ensure consistencyamong various formulations. As shown in FIG. 25, both the storage moduli(G′) and loss moduli (G″) of the mixtures increased with time and therecorded values levelled off after 300 sec, suggesting that the SPAACcrosslinking was completed within a matter of minutes. The sol-geltransition point, defined as the point where G′ increased to across withG″, was not observed using this testing protocol, likely due to therapid occurrence of SPAAC crosslinking within the first minute of mixingthe respective macromers. Indeed, rigorous quantitative comparisons ofthe gelling rates among the various fast-gelling formulations would bechallenging without significant modification of the mixing and testingprotocols. Qualitative observation of the gelling process by tilting thevials upon mixing the respective macromers revealed that the gellingrate followed the following trend:4-arm-PEG10k-(DBCO)₄>PEG6k-(DBCO)₂>PEG20k-(DBCO)₂ upon mixing withPEG-P(AzDXO)_(2m). All formulations started to gel in less than 1 min bythe vial tilting test (FIG. 22d ), consistent with the implicationsderived from the time-sweep oscillatory rheology tests (FIG. 25). Such afast gelling rate will be beneficial for applications of the macromersas injectable hydrogel formulations to repair tissue defects where thecontainment of the hydrogel within the local environment is critical.

The equilibrium shear modulus of the hydrogel can be tuned by adjustingthe length between the reactive groups (PEG block length) or macromerstructures (linear vs. 4-arm). As shown in FIG. 25, at the same weightcontent (10 w/v %), hydrogels crosslinked from PEG-(DBCO)_(x) andPEG6k-P(AzDXO)₄ (solid symbols) exhibited higher storage moduli thanthose crosslinked from PEG-(DBCO)_(x) and PEG20k-P(AzDXO)₄ (opensymbols), suggesting that the storage modulus inversely correlated withthe PEG length between the P(AzDXO) blocks. Among all 6 formulations,the hydrogel crosslinked by PEG6k-P(AzDXO)₄ and 4-arm-PEG10k-(DBCO)₄exhibited the highest storage modulus throughout the gelling process,with its equilibrium G′ approaching 6.0 KPa. This is largely due to thehighest chemical crosslinking density accomplished by the 4-armed DBCOcrosslinker and the PEG-P(AzDXO)_(2m) with the shortest PEG blocklength. In addition to chemical crosslinking density, degree of physicalentanglement also played an important role in the storage modulus of thegel, especially when both macromers contain sufficiently long PEGblocks. For instance, the gel crosslinked by PEG20k-P(AzDXO)₄ andPEG20k-(DBCO)₂ exhibited higher modulus than that crosslinked byPEG20k-P(AzDXO)₄ and PEG6k-(DBCO)₂. Overall, the equilibrium shearmoduli of the 6 gel systems decrease in the following order:PEG6k-P(AzDXO)₄+4-arm-PEG10k-(DBCO)₄ (6KPa)>PEG6k-P(AzDXO)₄+PEG6k-(DBCO)₂ (3.5kPa)>PEG6k-P(AzDXO)₄+PEG20k-(DBCO)₂ (2.7kPa)>PEG20k-P(AzDXO)₄+4-arm-PEG10k-(DBCO)₄ (1.3kPa)>PEG20k-P(AzDXO)₄+PEG20k-(DBCO)₄ (0.7kPa)>PEG20k-P(AzDXO)₄+PEG6k-(DBCO)₂ (0.5 kPa).

The cytocompatibility of PEG-P(AzDXO)_(2m) and PEG-(DBCO)_(x) macromersand the respective “click” hydrogels were evaluated in vitro. Bonemarrow-derived stromal cells (BMSC) cultured in the presence of 10 w/v %of each macromer showed comparable cell viability at 48 h to thosecultured without any macromer supplements (FIG. 28), supportingexcellent cytocompatibility of these macromers. Further, we showed thatmost BMSCs encapsulated by “clicking” the macromer components (FIG. 22d) remained viable, as supported by the dominant green fluorescent stainsfor live cells observed at 24 h upon performing a live/dead cellstaining on the cell-hydrogel constructs (FIG. 26a ). No statisticallysignificant difference in the hydrogel storage modulus was detected uponthe encapsulation of BMSC in any of the formulations investigated.

MTT cell viability assay performed on the hydrogel-cell constructs 48 hafter cell encapsulation (FIG. 26b ) showed that BMSC cells encapsulatedin all “click” hydrogels (10⁶ cells/mL) exhibited higher viability thanthose photo-encapsulated in the PEG6k-DMA hydrogel that was widely usedfor cell encapsulation in cartilage engineering. (Bryant, et al. J.Biomed. Mater. Res. 2002, 59, 63; Elisseeff, et al. J. Biomed. Mater.Res. 2000, 51, 164.) In our hands, the gellation of the 10 w/v %PEG6k-DMA gels in the presence of BMSC (10⁶ cells/mL) and 0.05 w/v %Irgacure-2959 photoinitiator required irradiation at 365 nm for 10 min.It is known that environmental conditions such as oxygen level couldlead to variation in the required polymerization time. The consistentlymore rapid gelling (within 1 min for most formulations) enabled by theSPAAC crosslinking presented here, coupled with the eliminated need fortoxic photoinitiators or UV irradiation, presents a significantadvantage.

Experimental Section

Chemicals.

Azido-functionalized cyclic carbonate monomer (AzDXO) was synthesized asdescribed previously. (Xu, et al. Macromolecules, 44, 2660.)Poly(ethylene glycol)diol (PEG, M_(n)=6,000, 10,000, 20,000 g/mol,Aldrich) and 4-arm-PEG (Mn=10,000 g/mol, JenKem Technolgoy) were driedunder vacuum in melt state for 3 h prior to use.1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) was purified by distillationwith calcium hydride under reduced pressure. Dichloromethane andchloroform were dried by distillation over P₂O₅ immediately prior touse. All other chemicals were used as received.

Synthesis of PEG-P(AzDXO)_(2m)

PEG-P(AzDXO)_(2n), macromers were prepared by initiating ROP of AzDXOwith PEG6k, PEG10k or PEG20k under the catalysis of DBU at rt in CH₂Cl₂.The AzDXO concentration and DBU concentration were kept at 0.1125 M and0.01 M, respectively. The amount of PEG was adjusted accordingly toobtained various copolymer compositions shown in Table 1. In arepresentative procedure for synthesizing PEG6k-P(AzDXO)₄, PEG6k (6.00g, 1.00 mmol) and AzDXO (0.955 g, 4.50 mmol) were dissolved in 38 mL ofCH₂Cl₂ under argon atmosphere. A 2-mL solution of DBU (0.2 M in CH₂Cl₂)was injected to initiate the polymerization. After 4 h, benzoic acid(0.122 g, 1.0 mmol) was added to neutralize DBU. The polymer waspurified by precipitation in 800 mL of ethyl ether. The precipitate wasthen redissolved in 40 mL of CH₂Cl₂ and precipitated again in 800 mL ofethyl ether, and repeated twice. The macromer was obtained as whitepowder and dried under vacuum at rt (6.740 g, yield=97%).

Synthesis of PEG-(DBCO)_(x).

The alkyne-containing macromers PEG-(DBCO)_(x) were synthesized byesterification of the hydroxyl ends of the respective linear PEG or4-arm-PEG with DBCO-acid. In a representative synthesis ofPEG-(DBCO)_(x) with high M_(n), PEG20k (4.0 g, ˜0.2 mmol) was dissolvedin 20 mL of chloroform. DBCO-acid (234.3 mg, 0.6 mmol),4-(dimethylamino)-pyridinium p-toluenesulfonate (DPTS, 29.4 mg, 0.1mmol) and N,N-diisopropylcarbodiimide (DIPC, 252.4 mg, 2.0 mmol) wereadded subsequently. After 10 h, 80 mL of chloroform was added and thesolution was washed by 20 mL of 0.1 M NaCl aqueous solution three times.The organic layer was dried with anhydrous sodium sulfate overnight.After filtering, the clear solution was dropped into 900 mL of ethylether, and the white precipitate was collected by filtration. The solidwas redissolved in 100 mL of chloroform and reprecipitated in 900 mL ofethyl ether and repeated twice until no residue catalysts could bedetected by ¹H NMR. The macromer was obtained as white powder and driedunder vacuum at rt (4.05 g, yield=97.4%).

NMR and GPC. ¹H (400 MHz) and ¹³C NMR (100 MHz) spectra were recorded ona Varian INOVA-400 spectrometer in deuterated chloroform (CDCl₃, 99.8atom % D with 0.03% v/v TMS). GPC measurements were taken on a VarianProStar HPLC system equipped with two 5-mm PLGel MiniMIX-D columns(Polymer Laboratory, Amherst, Mass.), a UV-vis detector and a PL-ELS2100evaporative light scattering detector (Polymer Laboratory, Amherst,Mass.). THF was used as an eluent at a flow rate of 0.3 mL/min at rt.The number-averaged molecular weight (M_(n)) and the polydispersityindex (PDI) were calculated by a Cirrus AIA GPC Software using narrowlydispersed polystyrenes (ReadyCal kits, PSS Polymer Standards ServiceInc. Germany) as calibration standards.

Rheology.

Dynamic rheology test was performed on an AR-2000 rheometer (TAInstruments) equipped with 20-mm parallel plates and a Peltier heatingunit. The gelling process of the various formulations and the evolutionof the shear modulus of the hydrogels were studied by oscillatory timesweep rheology experiments at 37° C. Aqueous solutions of PEG-P(AzDXO)₂mand PEG-(DBCO)_(x) (10 w/v %) in cell expansion media (α-MEM withoutascorbic acid, 20% FBS) with 1:1 molar ratio of the azide groups to thealkyne groups were loaded on the bottom plate sequentially and mixed bypipette. The mixed solution was kept between the parallel plates for 60sec before the experiment and data collection were initiated to ensureconsistency among various formulations. An oscillatory frequency of 1 Hzand a strain of 0.5% were applied.

Bone Marrow Stromal Cell (BMSC) Encapsulations.

PEG-P(AzDXO)_(2m) and PEG-(DBCO)_(x) were dissolved in BMSC expansionmedia to reach a 10 w/v % concentration, respectively. The solutionswere sterile-filtered through a 0.22-μm filter. BMSC were harvested fromthe femur and tibia of skeletally mature male rats (Charles River SASCOSD) and enriched by adherent culture as previously described. (Song, etal. J. Biomed. Mater. Res., Part A 2009, 89A, 1098.) Passage 1 BMSCcells were plated overnight in expansion media, trypsinized, counted andsuspended into the respective macromer solutions (10⁶ cells/mL). The twoBMSC-macromer solutions, PEG-P(AzDXO)_(2m) and PEG-(DBCO)_(x), weremixed in a total volume of 50 μL in 96-well tissue culture plate. Anextra 200 μL of expansion media was added to each well after 45 min. Asa control hydrogel for BMSC encapsulation, poly(ethyleneglycol)dimethylacrylate (PEGDMA Mn˜6000 g/mol) was alsophoto-crosslinked in the presence of BMSC cells. PEGDMA (10 w/v %) andthe photo initiator Irgacure-2959 (0.05 w/v %) were dissolved in PBS (pH7.4) or BMSC expansion media. The passage 1 BMSC cells were suspended in50 μL of PEGDMA/Irgacure-2959 solution (10⁶ cells/mL) and irradiatedwith 365 nm UV light for 10 min. An extra 200 μL of expansion media wasadded to each well immediately after the photo-polymerization. Allcell-hydrogel constructs were cultured for 24 and 48 h in a humidifiedincubation (5% CO₂, 37° C.) before being subjected to live/dead cellstaining or MTT cell viability assay. A sample size of 3 was applied toall cell-hydrogel constructs cultured for MTT.

Live and Dead Cell Staining of the Hydrogel-BMSC Constructs.

The hydrogel-cell constructs were stained using a LIVE/DEAD®viability/cytotoxicity kit (Molecular Probes) according the vendor'sprotocol. Living cells will be stained with green fluorescence byintracellular esterase catalyzed hydrolysis of Calcein AM, and deadcells will be stained red by Ethidium homodimer-1 after penetratingthrough the damaged membranes and binding of with nucleic acids. Thestained hydrogel-cell construct was mounted on microscope slide andimaged by a Leica SP laser scanning confocal microscope. ConfocalZ-stack images of encapsulated BMSC cells over the depth of 400 μm (20consecutive 20-μm slices) were overlaid.

MTT Cell Viability Assay.

The viability of the MBSC cells cultured on tissue culture plate in thepresence of 10 w/v % macromers or those encapsulated in 3-D hydrogelswere evaluated using MTT cell proliferation kit (Roche) after 48-hculture in expansion media (α-MEM without ascorbic acid, 20% FBS) in96-well plates. To a total volume of 150 μl of culture media andcell-hydrogel construct, 15 μl of MTT labelling reagent was added andincubated for 8 h at 37° C. on an orbital shaker. A 150-μlsolubilization solution was then added to each well, and incubated at37° C. on the orbital shaker for 36 h to fully dissolve and release thepurple formazan crystals from the 3-D hydrogels. The absorbance at 571nm was read on a MULTISCANFC spectrophotometer (Thermo Scientific). Asample size of 3 was applied to each construct or culture condition.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

The representative examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the examples andthe references to the scientific and patent literature included herein.The examples contain important additional information, exemplificationand guidance which can be adapted to the practice of this invention inits various embodiments and equivalents thereof.

What is claimed is:
 1. A compound having the structural formula of:

wherein each of R₁, R₂, R₅ and R₆ is H; and each of R₃ and R₄ is—(CH₂)_(p)—N₃, wherein p is an integer from about 1 to about
 16. 2. Thecompound of claim 1, wherein p=1.